RNAi Inhibition of Alpha-ENaC Expression

ABSTRACT

The invention relates to compositions and methods for modulating the expression of alpha-ENaC, and more particularly to the downregulation of alpha-ENaC expression by chemically modified oligonucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 16/752,325, filed Jan. 24, 2020, which is a divisional of U.S. patent application Ser. No. 15/874,396, filed Jan. 18, 2018, now U.S. patent Ser. No. 10/544,418, which is a continuation of U.S. patent application Ser. No. 15/260,824, filed on Sep. 9, 2016, now U.S. Pat. No. 9,914,927, which is a continuation of U.S. patent application Ser. No. 14/729,104, filed on Jun. 3, 2015, now U.S. Pat. No. 9,476,052, which is a divisional of U.S. patent application Ser. No. 12/683,146, filed on Jan. 6, 2010, now U.S. Pat. No. 9,074,212, which is a divisional of U.S. patent application Ser. No. 12/140,112, filed on Jun. 16, 2008, now U.S. Pat. No. 7,718,632, which claims priority to EP Application No. 07110376.6, filed Jun. 15, 2007 and EP Application No. 07114265.7, filed Aug. 13, 2007, the contents of each of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to the field of ENaC-mediated airway ion transport and compositions and methods for modulating alpha-ENaC expression, and more particularly to the down-regulation of alpha-ENaC by oligonucleotides via RNA interference which are administered locally to the lungs and nasal passage via inhalation/intranasal administration, or are administered systemically, e.g. by via intravenous injection.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., Nature 391:806-811, 1998). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. This technology has been reviewed numerous times recently, see, for example Novina, C. D: and Sharp, P., Nature 2004, 430:161, and Sandy, P., et al., Biotechniques 2005, 39:215, hereby incorporated by reference.

The mucosal surfaces at the interface between the environment and the body have evolved a number of protective mechanisms. A principal form of such innate defense is to cleanse these surfaces with liquid. Typically, the quantity of the liquid layer on a mucosal surface reflects the balance between epithelial liquid secretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupled with water (and a cation counter-ion), and epithelial liquid absorption, often reflecting Na⁺ absorption, coupled with water and counter anion (Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by too little protective liquid on those mucosal surfaces created by an imbalance between secretion (too little) and absorption (relatively too much). The defective salt transport processes that characterize these mucosal dysfunctions reside in the epithelial layer of the mucosal surface. One approach to replenish the protective liquid layer on mucosal surfaces is to “re-balance” the system by blocking Na⁺ channel mediated liquid absorption. The epithelial protein that mediates the rate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺ channel (ENaC). Alpha-ENaC is positioned on the apical surface of the epithelium, i.e. the mucosal surface-environmental interface. Inhibition of alpha-ENaC mediated Na⁺ mediated liquid absorption may achieve therapeutic utility. Therefore, there is a need for the development of effective therapies for the treatment and prevention of diseases or disorders in which alpha-ENaC is implicated, e.g. cystic fibrosis in humans and animals, and particularly for therapies with high efficiency. One prerequisite for high efficiency is that the active ingredient is not degraded too quickly in a physiological environment.

SUMMARY

The present invention provides specific compositions and methods that are useful in reducing alpha-ENaC levels in a subject, e.g., a mammal, such as a human, e.g. by inhaled, intranasal or intratracheal administration of such agents.

The present invention specifically provides iRNA agents consisting of, consisting essentially of or comprising at least 15 or more contiguous nucleotides for alpha-ENaC, and more particularly agents comprising 15 or more contiguous nucleotides from one of the sequences provided in Tables 1A-1D. The iRNA agent preferably comprises less than 30 nucleotides per strand, e.g., 21-23 nucleotides, such as those provided in Tables 1A-1D. The double stranded iRNA agent can either have blunt ends or more preferably have overhangs of 1-4 nucleotides from one or both 3′ ends of the agent.

Further, the iRNA agent can either contain only naturally occurring ribonucleotide subunits, or can be synthesized so as to contain one or more modifications to the sugar, phosphate or base of one or more of the ribonucleotide subunits that is included in the agent. The iRNA agent can be further modified so as to be attached to a ligand that is selected to improve stability, distribution or cellular uptake of the agent, e.g. cholesterol. The iRNA agents can further be in isolated form or can be part of a pharmaceutical composition used for the methods described herein, particularly as a pharmaceutical composition formulated for delivery to the lungs or nasal passage or formulated for parental administration. The pharmaceutical compositions can contain one or more iRNA agents, and in some embodiments, will contain two or more iRNA agents, each one directed to a different segment the alpha-ENaC gene.

One aspect of the present invention relates to a double-stranded oligonucleotide comprising at least one non-natural nucleobase. In certain embodiments, the non-natural nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a preferred embodiment, the non-natural nucleobase is difluorotolyl. In certain embodiments, only one of the two oligonucleotide strands comprising the double-stranded oligonucleotide contains a non-natural nucleobase. In certain embodiments, both of the oligonucleotide strands comprising the double-stranded oligonucleotide independently contain a non-natural nucleobase.

The present invention further provides methods for reducing the level of alpha-ENaC mRNA in a cell. Such methods comprise the step of administering one of the iRNA agents of the present invention to a subject as further described below. The present methods utilize the cellular mechanisms involved in RNA interference to selectively degrade the target RNA in a cell and are comprised of the step of contacting a cell with one of the iRNA agents of the present invention. Such methods can be performed directly on a cell or can be performed on a mammalian subject by administering to a subject one of the iRNA agents/pharmaceutical compositions of the present invention. Reduction of target RNA in a cell results in a reduction in the amount of encoded protein produced, and in an organism, results in reduction of epithelial potential difference, decreased fluid absorption and increased mucociliary clearance.

The methods and compositions of the invention, e.g., the methods and iRNA agent compositions can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from this description, the drawings, and from the claims. This application incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Restriction digest map of pXoon construct for cloned cynomolgous α-EnaC.

FIG. 2: Cloning of the predicted off-target and the on-target recognition sites into the AY535007 dual luciferase reporter construct. Fragments consist of 19 nt of the predicted target site and 10 nt of flanking sequence at both the 5′ and 3′ ends.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

For ease of exposition the term “nucleotide” or “ribonucleotide” is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood that the usage of the term “ribonucleotide” or “nucleotide” herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety, as further described below, at one or more positions.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, each of which is described herein or is well known in the RNA synthetic art. While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those that have a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′-modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as used herein, is an RNA agent, which can downregulate the expression of a target gene, e.g. ENaC gene SCNN1A. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms.

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”), as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interstrand hybridization can form a region of duplex structure. A “strand” herein refers to a contiguous sequence of nucleotides (including non-naturally occurring or modified nucleotides). The two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g., by a linker, e.g., a polyethyleneglycol linker, to form one molecule. At least one strand can include a region which is sufficiently complementary to a target RNA. Such strand is termed the “antisense strand.” A second strand of the dsRNA agent, which comprises a region complementary to the antisense strand, is termed the “sense strand.” However, a ds iRNA agent can also be formed from a single RNA molecule which is at least partly self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region. The latter are herein referred to as short hairpin RNAs or shRNAs. In such case, the term “strand” refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule.

Although, in mammalian cells, long ds iRNA agents can induce the interferon response which is frequently deleterious, short ds iRNA agents do not trigger the interferon response, at least not to an extent that is deleterious to the cell and/or host (Manche et al., Mol. Cell. Biol. 12:5238, 1992; Lee et al., Virology 199:491, 1994; Castelli et al., J. Exp. Med. 186:967, 1997; Zheng et al., RNA 10:1934, 2004; Heidel et al., Nature Biotechnol. 221579). The iRNA agents of the present invention include molecules which are sufficiently short that they do not trigger a deleterious non-specific interferon response in normal mammalian cells. Thus, the administration of a composition including an iRNA agent (e.g., formulated as described herein) to a subject can be used to decrease expression of alpha-ENaC in the subject, while circumventing an interferon response. Molecules that are short enough that they do not trigger a deleterious interferon response are termed siRNA agents or siRNAs herein. “siRNA agent” or “siRNA” as used herein, refers to an iRNA agent, e.g., a ds iRNA agent, that is sufficiently short that it does not induce a deleterious interferon response in a mammalian, and particularly a human, cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs.

The isolated iRNA agents described herein, including ds iRNA agents and siRNA agents, can mediate the decreased expression of alpha-ENaC, e.g., by RNA degradation. For convenience, such RNA is also referred to herein as the RNA to be silenced. Such a nucleic acid is also referred to as a “target RNA”, sometimes “target RNA molecule” or sometimes “target gene”.

As used herein, the phrase “mediates RNAi” refers to the ability of an agent to silence, in a sequence-specific manner, a target gene. “Silencing a target gene” means the process whereby a cell containing and/or expressing a certain product of the target gene when not in contact with the agent, will contain and/or express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent. Such product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.

As used herein, the term “complementary” is used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule, e.g., alpha-ENaC mRNA. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ from the target sequences by at least 2, 3 or 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA (e.g., alpha-ENaC mRNA) if the iRNA agent reduces the production of a protein encoded by the target RNA in a cell. The iRNA agent may also be “exactly complementary” to the target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” iRNA agent can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target alpha-ENaC RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementarity is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference. Preferred iRNA agents will be based on or consist of or comprise the sense and antisense sequences provided in Tables 1A-1D.

As used herein, “essentially identical” when used referring to a first nucleotide sequence in comparison to a second nucleotide sequence means that the first nucleotide sequence is identical to the second nucleotide sequence except for up to one, two or three nucleotide substitutions (e.g., adenosine replaced by uracil). “Essentially retaining the ability to inhibit alpha-ENaC expression in cultured human cells,” as used herein referring to an iRNA agent not identical to but derived from one of the iRNA agents of Tables 1A-1D by deletion, addition or substitution of nucleotides, means that the derived iRNA agent possesses an inhibitory activity not less than 20% of the inhibitory activity of the iRNA agent of Tables 1A-1D from which it was derived. For example, an iRNA agent derived from an iRNA agent of Tables 1A-1D which lowers the amount of alpha-ENaC mRNA present in cultured human cells by 70% may itself lower the amount of mRNA present in cultured human cells by at least 50% in order to be considered as essentially retaining the ability to inhibit alpha-ENaC replication in cultured human cells. Optionally, an iRNA agent of the invention may lower the amount of alpha-ENaC mRNA present in cultured human cells by at least 50%.

As used herein, a “subject” refers to a mammalian organism undergoing treatment for a disorder mediated by alpha-ENaC. The subject can be any mammal, such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. In the preferred embodiment, the subject is a human.

Design and Selection of iRNA Agents

As used herein, “disorders associated with alpha-ENaC expression” refers to any biological or pathological state that (1) is mediated at least in part by the presence of alpha-ENaC and (2) whose outcome can be affected by reducing the level of the alpha-ENaC present. Specific disorders associated with alpha-ENaC expression are noted below.

The present invention is based on the design, synthesis and generation of iRNA agents that target alpha-ENaC and the demonstration of silencing of the alpha-ENaC gene in vitro in cultured cells after incubation with an iRNA agent, and the resulting protective effect towards alpha-ENaC mediated disorders.

An iRNA agent can be rationally designed based on sequence information and desired characteristics. For example, an iRNA agent can be designed according to the relative melting temperature of the candidate duplex. Generally, the duplex should have a lower melting temperature at the 5′ end of the antisense strand than at the 3′ end of the antisense strand.

The present invention provides compositions containing siRNA(s) and/or shRNA(s) targeted to one or more alpha-ENaC transcripts.

For any particular gene target that is selected, the design of siRNAs or shRNAs for use in accordance with the present invention will preferably follow certain guidelines. Also, in many cases, the agent that is delivered to a cell according to the present invention may undergo one or more processing steps before becoming an active suppressing agent (see below for further discussion); in such cases, those of ordinary skill in the art will appreciate that the relevant agent will preferably be designed to include sequences that may be necessary for its processing.

Diseases mediated by dysfunction of the epithelial sodium channel, include diseases associated with the regulation of fluid volumes across epithelial membranes. For example, the volume of airway surface liquid is a key regulator of mucociliary clearance and the maintenance of lung health. The blockade of the epithelial sodium channel will promote fluid accumulation on the mucosal side of the airway epithelium thereby promoting mucus clearance and preventing the accumulation of mucus and sputum in respiratory tissues (including lung airways). Such diseases include respiratory diseases, such as cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, respiratory tract infections (acute and chronic; viral and bacterial) and lung carcinoma. Diseases mediated by blockade of the epithelial sodium channel also include diseases other than respiratory diseases that are associated with abnormal fluid regulation across an epithelium, perhaps involving abnormal physiology of the protective surface liquids on their surface, e.g., xerostomia (dry mouth) or keratoconjunctivitis sire (dry eye). Furthermore, blockade of the epithelial sodium channel in the kidney could be used to promote diuresis and thereby induce a hypotensive effect.

Treatment in accordance with the invention may be symptomatic or prophylactic.

Asthma includes both intrinsic (non-allergic) asthma and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchitic asthma, exercise-induced asthma, occupational asthma and asthma induced following bacterial infection. Treatment of asthma is also to be understood as embracing treatment of subjects, e.g., of less than 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed or diagnosable as “wheezy infants”, an established patient category of major medical concern and now often identified as incipient or early-phase asthmatics. (For convenience this particular asthmatic condition is referred to as “wheezy-infant syndrome”.)

Prophylactic efficacy in the treatment of asthma will be evidenced by reduced frequency or severity of symptomatic attack, e.g., of acute asthmatic or bronchoconstrictor attack, improvement in lung function or improved airways hyperreactivity. It may further be evidenced by reduced requirement for other, symptomatic therapy, i.e., therapy for or intended to restrict or abort symptomatic attack when it occurs, e.g., anti-inflammatory (e.g., corticosteroid) or bronchodilatory. Prophylactic benefit in asthma may, in particular, be apparent in subjects prone to “morning dipping”. “Morning dipping” is a recognized asthmatic syndrome, common to a substantial percentage of asthmatics and characterized by asthma attack, e.g., between the hours of about 4-6 am, i.e., at a time normally substantially distant from any previously administered symptomatic asthma therapy.

Chronic obstructive pulmonary disease includes chronic bronchitis or dyspnea associated therewith, emphysema, as well as exacerbation of airways hyperreactivity consequent to other drug therapy, in particular, other inhaled drug therapy. The invention is also applicable to the treatment of bronchitis of whatever type or genesis including, e.g., acute, arachidic, catarrhal, croupus, chronic or phthinoid bronchitis.

Based on the results shown herein, the present invention provides iRNA agents that reduce alpha-ENaC expression in cultured cells and in a subject, e.g. a mammalian, for example a human. Tables 1A-1D provide exemplary iRNA agents targeting alpha-ENaC, based on the standard nomenclature abbreviations given in Table A.

Table 1A, Seq Id No.s 305-608, Table 1B and Table 1D, Seq Id No.s 1519-1644 list siRNAs that do not comprise nucleotide modifications except for one phosphorothioate linkage between the 3′-terminal and the penultimate thymidines. The remaining Seq Ids in Tables 1A-1D lists siRNAs wherein all nucleotides comprising pyrimidine bases are 2′-O-methyl-modified nucleotides in the sense strand, and all uridines in a sequence context of 5′-ua-3′ as well as all cytidines in a sequence context of or 5′-ca-3′ are 2′-O-methyl-modified nucleotides in the antisense strand.

Based on these results, the invention specifically provides an iRNA agent that includes a sense strand having at least 15 contiguous nucleotides of the sense strand sequences of the agents provided in Tables 1A-1D, and an antisense strand having at least 15 contiguous nucleotides of the antisense sequences of the agents provided in Tables 1A-1D.

The iRNA agents shown in Tables 1A-1D are composed of two strands of 19 nucleotides in length which are complementary or identical to the target sequence, plus a 3′-TT overhang. The present invention provides agents that comprise at least 15, or at least 16, 17, or 18, or 19 contiguous nucleotides from these sequences. However, while these lengths may potentially be optimal, the iRNA agents are not meant to be limited to these lengths. The skilled person is well aware that shorter or longer iRNA agents may be similarly effective, since, within certain length ranges, the efficacy is rather a function of the nucleotide sequence than strand length. For example, Yang, et al., PNAS 99:9942-9947 (2002), demonstrated similar efficacies for iRNA agents of lengths between 21 and 30 base pairs. Others have shown effective silencing of genes by iRNA agents down to a length of approx. 15 base pairs (Byrom, et al., “Inducing RNAi with siRNA Cocktails Generated by RNase III” Tech Notes 10(1), Ambion, Inc., Austin, Tex.).

Therefore, it is possible and contemplated by the instant invention to select from the sequences provided in Tables 1A-1D a partial sequence of between 15 to 19 nucleotides for the generation of an iRNA agent derived from one of the sequences provided in Tables 1A-1D. Alternatively, one may add one or several nucleotides to one of the sequences provided in Tables 1A-1D, or an agent comprising 15 contiguous nucleotides from one of these agents, preferably, but not necessarily, in such a fashion that the added nucleotides are complementary to the respective sequence of the target gene, e.g., alpha-ENaC. For example, the first 15 nucleotides from one of the agents can be combined with the 8 nucleotides found 5′ to these sequence in alpha-ENaC mRNA to obtain an agent with 23 nucleotides in the sense and antisense strands. All such derived iRNA agents are included in the iRNA agents of the present invention, provided they essentially retain the ability to inhibit alpha-ENaC replication in cultured human cells.

The antisense strand of an iRNA agent should be equal to or at least, 14, 15, 16, 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 60, 50, 40, or 30, nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The sense strand of an iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 60, 50, 40, or 30 nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double stranded portion of an iRNA agent should be equal to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50 nucleotide pairs in length. It should be equal to or less than 60, 50, 40, or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length.

Generally, the iRNA agents of the instant invention include a region of sufficient complementarity to the alpha-ENaC mRNA, and are of sufficient length in terms of nucleotides, that the iRNA agent, or a fragment thereof, can mediate down regulation of the alpha-ENaC gene. It is not necessary that there be perfect complementarity between the iRNA agent and the target gene, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of an alpha-ENaC mRNA.

Therefore, the iRNA agents of the instant invention include agents comprising a sense strand and antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical, as defined below, to one of the sequences of Tables 1A-1D, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit alpha-ENaC expression in cultured human cells. These agents will therefore possess at least 15 nucleotides identical to one of the sequences of Tables 1A-1D, but 1, 2 or 3 base mismatches with respect to either the target alpha-ENaC sequence or between the sense and antisense strand are introduced. Mismatches to the target alpha-ENaC RNA sequence, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5′ and/or 3′ terminus, most preferably within 6, 5, 4, or 3 nucleotides of the 5′-terminus of the sense strand or the 3′-terminus of the antisense strand. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double stranded character of the molecule.

It is preferred that the sense and antisense strands be chosen such that the iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, an iRNA agent contains sense and antisense strands, preferably paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred siRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 to 4, or preferably 2 or 3 nucleotides, in length, at one or both ends of the iRNA agent. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The unpaired nucleotides forming the overhang can be ribonucleotides, or they can be deoxyribonucleotides, preferably thymidine. 5′-ends are preferably phosphorylated, or they may be unphosphorylated.

Preferred lengths for the duplexed region are between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the siRNA agent range discussed above. siRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the siRNA agent are linked, e.g., covalently linked, are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3′ overhang are also within the invention.

Evaluation of Candidate iRNA Agents

As noted above, the present invention provides a system for identifying siRNAs that are useful as inhibitors of alpha-ENaC. Since, as noted above, shRNAs are processed intracellularly to produce siRNAs having duplex portions with the same sequence as the stem structure of the shRNA, the system is equally useful for identifying shRNAs that are useful as inhibitors of alpha-ENaC. For purposes of description this section will refer to siRNAs, but the system also encompasses corresponding shRNAs. Specifically, the present invention demonstrates the successful preparation of siRNAs targeted to inhibit alpha-ENaC activity. The techniques and reagents described herein can readily be applied to design potential new siRNAs, targeted to other genes or gene regions, and tested for their activity in inhibiting alpha-ENaC as discussed herein.

In various embodiments of the invention potential alpha-ENaC inhibitors can be tested for suppression of endogenous alpha-ENaC expression by introducing candidate siRNA(s) into cells (e.g., by exogenous administration or by introducing a vector or construct that directs endogenous synthesis of siRNA into the cell), or in laboratory animals by pulmonary or nasal administration. Alternately, potential alpha-ENaC inhibitors can be tested in vitro by transient co-transfection of candidate siRNA(s) together with an alpha-ENaC-expression plasmid. The ability of the candidate siRNA(s) to reduce target transcript levels and/or to inhibit or suppress one or more aspects or features of alpha-ENaC activity such as epithelial potential difference or airway surface fluid absorption is then assessed.

Cells or laboratory animals to which inventive siRNA compositions have been delivered (test cells/animals) may be compared with similar or comparable cells or laboratory animals that have not received the inventive composition (control cells/animals, e.g., cells/animals that have received either no siRNA or a control siRNA such as an siRNA targeted to a non-endogenous transcript such as green fluorescent protein (GFP)). The ion transport phenotype of the test cells/animals can be compared with the phenotype of control cells/animals, providing that the inventive siRNA share sequence cross-reactivity with the test cell type/species. Production of alpha-ENaC protein and short circuit current (in vitro or ex vivo) may be compared in the test cells/animals relative to the control cells/animals. Other indicia of alpha-ENaC activity, including ex vivo epithelial potential difference or in vivo mucocilliary clearance or whole body magnetic resonance imaging (MRI), can be similarly compared. Generally, test cells/animals and control cells/animals would be from the same species and, for cells, of similar or identical cell type. For example, cells from the same cell line could be compared. When the test cell is a primary cell, typically the control cell would also be a primary cell.

For example, the ability of a candidate siRNA to inhibit alpha-ENaC activity may conveniently be determined by (i) delivering the candidate siRNA to cells (ii) assessing the expression levels of alpha-ENaC mRNA relative to an endogenously expressed control gene (iii) comparing the amiloride-sensitive current in an in vitro cell model produced in the presence of the siRNA with the amount produced in the absence of the siRNA. This latter assay may be used to test siRNAs that target any target transcript that may influence alpha-ENaC activity indirectly and is not limited to siRNAs that target the transcripts that encode the ENaC channel subunits.

The ability of a candidate siRNA to reduce the level of the target transcript may be assessed by measuring the amount of the target transcript using, for example, Northern blots, nuclease protection assays, probe hybridization, reverse transcription (RT)-PCR, real-time RT-PCR, microarray analysis, etc. The ability of a candidate siRNA to inhibit production of a polypeptide encoded by the target transcript (either at the transcriptional or post-transcriptional level) may be measured using a variety of antibody-based approaches including, but not limited to, Western blots, immunoassays, ELISA, flow cytometry, protein microarrays, etc. In general, any method of measuring the amount of either the target transcript or a polypeptide encoded by the target transcript may be used.

In general, certain preferred alpha-ENaC iRNA inhibitors reduce the target transcript level at least about 2 fold, preferably at least about 4 fold, more preferably at least about 8 fold, at least about 16 fold, at least about 64 fold or to an even greater degree relative to the level that would be present in the absence of the inhibitor (e.g., in a comparable control cell lacking the inhibitor). In general, certain preferred alpha-ENaC iRNA inhibitors inhibit ENaC channel activity, so that the activity is lower in a cell containing the inhibitor than in a control cell not containing the inhibitor by at least about 2 fold, preferably at least about 4 fold, more preferably at least about 8 fold, at least about 16 fold, at least about 64 fold, at least about 100 fold, at least about 200 fold, or to an even greater degree.

Certain preferred alpha-ENaC iRNA inhibitors inhibit ENaC channel activity for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours or at least 168 hours following administration of the siRNA and infection of the cells. Certain preferred alpha-ENaC inhibitors prevent (i.e., reduce to undetectable levels) or significantly reduce alpha-ENaC activity for at least 24 hours, at least 36 hours, at least 48 hours, or at least 60 hours following administration of the siRNA. According to various embodiments of the invention a significant reduction in alpha-ENaC activity is a reduction to less than approximately 90% of the level that would occur in the absence of the siRNA, a reduction to less than approximately 75% of the level that would occur in the absence of the siRNA, a reduction to less than approximately 50% of the level that would occur in the absence of the siRNA, a reduction to less than approximately 25% of the level that would occur in the absence of the siRNA, or a reduction to less than approximately 10% of the level that would occur in the absence of the siRNA. Reduction in alpha-ENaC activity may be measured using any suitable method including, but not limited to, short circuit current measurement of amiloride sensitivity in vitro, epithelial potential difference ex vivo or in vivo mucocilliary clearance or whole body/lung MRI.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to stability, e.g., its susceptibility to cleavage by an endonuclease or exonuclease, such as when the iRNA agent is introduced into the body of a subject. Methods can be employed to identify sites that are susceptible to modification, particularly cleavage, e.g., cleavage by a component found in the body of a subject. Such methods may include the isolation and identification of most abundant fragments formed by degradation of the candidate iRNA agent after its incubation with isolated biological media in vitro, e.g. serum, plasma, sputum, cerebrospinal fluid, or cell or tissue homogenates, or after contacting a subject with the candidate iRNA agent in vivo, thereby identifying sites prone to cleavage. Such methods are, for example, without limitation, in International Patent Application Publication No. WO2005115481, filed on May 27, 2005.

When sites susceptible to cleavage are identified, a further iRNA agent can be designed and/or synthesized wherein the potential cleavage site is made resistant to cleavage, e.g. by introduction of a 2′-modification on the site of cleavage, e.g. a 2′-O-methyl group. This further iRNA agent can be retested for stability, and this process may be iterated until an iRNA agent is found exhibiting the desired stability.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting alpha-ENaC gene expression can be tested for functionality in vivo in an animal model (e.g., in a mammal, such as in mouse, rat, guinea-pig or primate). For example, the iRNA agent can be administered to an animal, and the iRNA agent evaluated with respect to its biodistribution, stability, and its ability to inhibit alpha-ENaC expression or modulate a biological or pathological process mediated at least in part by alpha-ENaC.

The iRNA agent can be administered directly to the target tissue, such as by injection, or the iRNA agent can be administered to the animal model in the same manner that it would be administered to a human. Preferably, the iRNA agent is delivered to the subject's airways, such as by intranasal, inhaled or intratracheal administration.

The iRNA agent can also be evaluated for its intracellular distribution. The evaluation can include determining whether the iRNA agent was taken up into the cell. The evaluation can also include determining the stability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNA agent in vivo can be facilitated by use of an iRNA agent conjugated to a traceable marker (e.g., a fluorescent marker such as fluorescein; a radioactive label, such as ³⁵S, ³²P, ³³P, or ³H; gold particles; or antigen particles for immunohistochemistry).

The iRNA agent can be evaluated with respect to its ability to down regulate alpha-alpha-ENaC expression. Levels of alpha-ENaC gene expression in vivo can be measured, for example, by in situ hybridization, or by the isolation of RNA from tissue prior to and following exposure to the iRNA agent. Where the animal needs to be sacrificed in order to harvest the tissue, an untreated control animal will serve for comparison. alpha-ENaC RNA can be detected by any desired method, including but not limited to RT-PCR, northern blot, branched-DNA assay, or RNAase protection assay. Alternatively, or additionally, alpha-ENaC gene expression can be monitored by performing western blot analysis or immunostaining on tissue extracts treated with the iRNA agent.

Potential alpha-ENaC inhibitors can be tested using any variety of animal models that have been developed. Compositions comprising candidate siRNA(s), constructs or vectors capable of directing synthesis of such siRNAs within a host cell, or cells engineered or manipulated to contain candidate siRNAs may be administered to an animal. The ability of the composition to suppress alpha-ENaC expression and/or to modify ENaC-dependent phenotypes and/or lessen their severity relative to animals that have not received the potential alpha-ENaC inhibitor is assessed. Such models include, but are not limited to, murine, rat, guinea pig, sheep and non-human primate models for ENaC-dependent phenotypes, all of which are known in the art and are used for testing the efficacy of potential alpha-ENaC therapeutics.

Utilising the systems invented for identifying candidate therapeutic siRNA agents, suitable therapeutic agents are selected from Duplex identifiers ND-8302, ND-8332, ND-8348, ND-8356, ND-8357, ND-8373, ND-8381, ND-8396, ND-8450 and ND-8453, more suitably selected from ND-8356, ND-8357 and ND-8396.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., ds RNA agents that mediate RNAi to inhibit expression of the alpha-ENaC gene.

RNA agents discussed herein include otherwise unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. Nucleic Acids Res. 22: 2183-2196, 1994. Such rare or unusual RNAs, often termed modified RNAs (apparently because they are typically the result of a post-transcriptional modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of the above are discussed herein.

Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. It may be desirable to modify one or both of the antisense and sense strands of an iRNA agent. As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the non-linking oxygen of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most, cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. Similarly, a modification may occur on the sense strand, antisense strand, or both. In some cases, the sense and antisense strand will have the same modifications or the same class of modifications, but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it may be desirable to modify only one strand, e.g. the sense strand.

Two prime objectives for the introduction of modifications into iRNA agents is their stabilization towards degradation in biological environments and the improvement of pharmacological properties, e.g. pharmacodynamic properties, which are further discussed below. Other suitable modifications to a sugar, base, or backbone of an iRNA agent are described in PCT Application No. PCT/US2004/01193, filed Jan. 16, 2004. An iRNA agent can include a non-naturally occurring base, such as the bases described in PCT Application No. PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include a non-naturally occurring sugar, such as a non-carbohydrate cyclic carrier molecule. Exemplary features of non-naturally occurring sugars for use in iRNA agents are described in PCT Application No. PCT/US2004/11829, filed Apr. 16, 2003.

An iRNA agent can include an internucleotide linkage (e.g., the chiral phosphorothioate linkage) useful for increasing nuclease resistance. In addition, or in the alternative, an iRNA agent can include a ribose mimic for increased nuclease resistance. Exemplary internucleotide linkages and ribose mimics for increased nuclease resistance are described in PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

An iRNA agent can include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in U.S. application Ser. No. 10/916,185, filed on Aug. 10, 2004.

An iRNA agent can have a ZXY structure, such as is described in PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplary amphipathic moieties for use with iRNA agents are described in PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type. iRNA agents complexed to a delivery agent are described in PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

An iRNA agent can have non-canonical pairings, such as between the sense and antisense sequences of the iRNA duplex. Exemplary features of non-canonical iRNA agents are described in PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.

Enhanced Nuclease Resistance

An iRNA agent, e.g., an iRNA agent that targets alpha-ENaC, can have enhanced resistance to nucleases.

One way to increase resistance is to identify cleavage sites and modify such sites to inhibit cleavage, as described in U.S. Application No. 60/559,917, filed on May 4, 2004. For example, the dinucleotides 5′-ua-3′, 5′-ca-3′, 5′-ug-3′, 5′-uu-3′, or 5′-cc-3′ can serve as cleavage sites. In certain embodiments, all the pyrimidines of an iRNA agent carry a 2′-modification in either the sense strand, the antisense strand, or both strands, and the iRNA agent therefore has enhanced resistance to endonucleases. Enhanced nuclease resistance can also be achieved by modifying the 5′ nucleotide, resulting, for example, in at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide; at least one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; or at least one 5′-cytidine-cytidine-3′ (5′-cc-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, as described in International Application No. PCT/US2005/018931, filed on May 27, 2005. The iRNA agent can include at least 2, at least 3, at least 4 or at least 5 of such dinucleotides. In a particularly preferred embodiment, the 5′ nucleotide in all occurrences of the sequence motifs 5′-ua-3′ and 5′-ca-3′ in either the sense strand, the antisense strand, or both strands is a modified nucleotide. Preferably, the 5′ nucleotide in all occurrences of the sequence motifs 5′-ua-3′, 5′-ca-3′ and 5′-ug-3′ in either the sense strand, the antisense strand, or both strands is a modified nucleotide. More preferably, all pyrimidine nucleotides in the sense strand are modified nucleotides, and the 5′ nucleotide in all occurrences of the sequence motifs 5′-ua-3′ and 5′-ca-3′ in the antisense strand are modified nucleotides, or where the antisense strand does comprise neither of a 5′-ua-3′ and a 5′-ca-3′ motif, in all occurrences of the sequence motif 5′-ug-3′.

Preferably, the 2′-modified nucleotides include, for example, a 2′-modified ribose unit, e.g., the 2′-hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.

Preferred substitutents are 2′-methoxyethyl, 2′-OCH₃, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

The inclusion of furanose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An iRNA agent can be further modified by including a 3′ cationic group, or by inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. In another alternative, the 3′-terminus can be blocked with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′ exonucleolytic cleavage. While not being bound by theory, a 3′ conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3′-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3′-5′-exonucleases.

Nucleolytic cleavage can also be inhibited by the introduction of phosphate linker modifications, e.g., phosphorothioate linkages. Thus, preferred iRNA agents include nucleotide dimers enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at a nonbridging position normally occupied by oxygen. The heteroatom can be S, Se, Nr₂, or Br₃. When the heteroatom is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Modified phosphate linkages are particularly efficient in inhibiting exonucleolytic cleavage when introduced near the 5′- or 3′-terminal positions, and preferably the 5′-terminal positions, of an iRNA agent.

5′ conjugates can also inhibit 5′-3′ exonucleolytic cleavage. While not being bound by theory, a 5′ conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5′-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3′-5′-exonucleases.

An iRNA agent can have increased resistance to nucleases when a duplexed iRNA agent includes a single-stranded nucleotide overhang on at least one end. In preferred embodiments, the nucleotide overhang includes 1 to 4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment, the unpaired nucleotide of the single-stranded overhang that is directly adjacent to the terminal nucleotide pair contains a purine base, and the terminal nucleotide pair is a G-C pair, or at least two of the last four complementary nucleotide pairs are G-C pairs. In further embodiments, the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in an exemplary embodiment the nucleotide overhang is 5′-gc-3′. In preferred embodiments, the nucleotide overhang is on the 3′-end of the antisense strand. In one embodiment, the iRNA agent includes the motif 5′-cgc-3′ on the 3′-end of the antisense strand, such that a 2-nt overhang 5′-gc-3′ is formed.

Thus, an iRNA agent can include modifications so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRMs, or Nuclease Resistance promoting Monomers, the corresponding modifications as NRM modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC, or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent.

NRM modifications include some which can be placed only at the terminus and others which can go at any position. Some NRM modifications can inhibit hybridization so it is preferable to use them only in terminal regions, and preferable to not use them at the cleavage site or in the cleavage region of a sequence which targets a subject sequence or gene, particularly on the antisense strand. They can be used anywhere in a sense strand, provided that sufficient hybridization between the two strands of the ds iRNA agent is maintained. In some embodiments it is desirable to put the NRM at the cleavage site or in the cleavage region of a sense strand, as it can minimize off-target silencing.

In most cases, NRM modifications will be distributed differently depending on whether they are comprised on a sense or antisense strand. If on an antisense strand, modifications which interfere with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference). Cleavage of the target occurs about in the middle of a 20 or 21 nt antisense strand, or about 10 or 11 nucleotides upstream of the first nucleotide on the target mRNA which is complementary to the antisense strand. As used herein cleavage site refers to the nucleotides on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means the nucleotides within 1, 2, or 3 nucleotides of the cleavage site, in either direction.

Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sense or antisense strand.

Tethered Ligands

The properties of an iRNA agent, including its pharmacological properties, can be influenced and tailored, for example, by the introduction of ligands, e.g. tethered ligands. In addition, pharmacological properties of an iRNA agent can be improved by incorporating a ligand in a formulation of the iRNA agent when the iRNA agent either has or does have a tethered ligand.

A wide variety of entities, e.g., ligands, can be tethered to an iRNA agent or used as formulation conjugate or additive, e.g., to the carrier of a ligand-conjugated monomer subunit. Examples are described below in the context of a ligand-conjugated monomer subunit but that is only preferred, entities can be coupled at other points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly, via an intervening tether to the carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. The ligand or tethered ligand may be present on the ligand-conjugated monomer when the ligand-conjugated monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” ligand-conjugated monomer subunit after a “precursor” ligand-conjugated monomer subunit has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether, e.g., TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer subunit into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor ligand-conjugated monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor ligand-conjugated monomer subunit tether.

In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophilic molecules, lipids, lectins, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan, synthetic (eg Oligo Lactate 15-mer) and natural (eg low and medium molecular weight) polymers, mulm, cyclodextrin or hyaluronic acid), proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Other examples include folic acid or epithelial cell receptor ligands, such as transferin.

The ligand may be a naturally occurring or recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a thyrotropin, melanotropin, surfactant protein A, mucin carbohydrate, a glycosylated polyaminoacid, transferrin, bisphosphonate, polyglutamate, polyaspartate, or an Arg-Gly-Asp (RGD) peptide or RGD peptide mimetic.

Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g. low density lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA), or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose.

The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tetracyclin.

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., liver tissue, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include the B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase. The cell permeation agent can be linked covalently to the iRNA agent or be part of an iRNA-peptide complex.

5′-Phosphate Modifications

In preferred embodiments, iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications of the antisense strand include those which are compatible with RISC-mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).

The sense strand can be modified in order to inactivate the sense strand and prevent formation of an active RISC, thereby potentially reducing off-target effects. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykanen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage.

Non-Natural Nucleobases

Nitropyrrolyl and nitroindolyl are non-natural nucleobases that are members of a class of compounds known as universal bases. Universal bases are those compounds that can replace any of the four naturally occurring bases without substantially affecting the melting behavior or activity of the oligonucleotide duplex. In contrast to the stabilizing, hydrogen-bonding interactions associated with naturally occurring nucleobases, it is postulated that oligonucleotide duplexes containing 3-nitropyrrolyl nucleobases are stabilized solely by stacking interactions. The absence of significant hydrogen-bonding interactions with nitropyrrolyl nucleobases obviates the specificity for a specific complementary base. In addition, various reports confirm that 4-, 5- and 6-nitroindolyl display very little specificity for the four natural bases. Interestingly, an oligonucleotide duplex containing 5-nitroindolyl was more stable than the corresponding oligonucleotides containing 4-nitroindolyl and 6-nitroindolyl. Procedures for the preparation of 1-(2′-O-methyl-β-D-ribofuranosyl)-5-nitroindole are described in Gaubert, G.; Wengel, J. Tetrahedron Letters 2004, 45, 5629. Other universal bases amenable to the present invention include hypoxanthinyl, isoinosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, and structural derivatives thereof. For a more detailed discussion, including synthetic procedures, of nitropyrrolyl, nitroindolyl, and other universal bases mentioned above see Vallone et al., Nucleic Acids Research, 27(17):3589-3596 (1999); Loakes et al., J. Mol. Bio., 270:426-436 (1997); Loakes et al., Nucleic Acids Research, 22(20):4039-4043 (1994); Oliver et al., Organic Letters, Vol. 3(13):1977-1980 (2001); Amosova et al., Nucleic Acids Research, 25(10):1930-1934 (1997); Loakes et al., Nucleic Acids Research, 29(12):2437-2447 (2001); Bergstrom et al., J. Am. Chem. Soc., 117:1201-1209 (1995); Franchetti et al., Biorg. Med. Chem. Lett. 11:67-69 (2001); and Nair et al., Nucleosides, Nucleotides & Nucleic Acids, 20(4-7):735-738 (2001).

Difluorotolyl is a non-natural nucleobase that functions as a universal base. Difluorotolyl is an isostere of the natural nucleobase thymine. But unlike thymine, difluorotolyl shows no appreciable selectivity for any of the natural bases. Other aromatic compounds that function as universal bases and are amenable to the present invention are 4-fluoro-6-methylbenzimidazole and 4-methylbenzimidazole. In addition, the relatively hydrophobic isocarbostyrilyl derivatives 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl are universal bases which cause only slight destabilization of oligonucleotide duplexes compared to the oligonucleotide sequence containing only natural bases. Other non-natural nucleobases contemplated in the present invention include 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivates thereof. For a more detailed discussion, including synthetic procedures, of difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, and other non-natural bases mentioned above, see: Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc., 119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324 (1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Morales et al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J. Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem., 63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci., 94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans., 1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1:1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632 (2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No. 6,218,108.

Transport of iRNA Agents into Cells

Not wishing to be bound by any theory, the chemical similarity between cholesterol-conjugated iRNA agents and certain constituents of lipoproteins (e.g. cholesterol, cholesteryl esters, phospholipids) may lead to the association of iRNA agents with lipoproteins (e.g. LDL, HDL) in blood and/or the interaction of the iRNA agent with cellular components having an affinity for cholesterol, e.g. components of the cholesterol transport pathway. Lipoproteins as well as their constituents are taken up and processed by cells by various active and passive transport mechanisms, for example, without limitation, endocytosis of LDL-receptor bound LDL, endocytosis of oxidized or otherwise modified LDLs through interaction with Scavenger receptor A, Scavenger receptor B1-mediated uptake of HDL cholesterol in the liver, pinocytosis, or transport of cholesterol across membranes by ABC (ATP-binding cassette) transporter proteins, e.g. ABC-A1, ABC-G1 or ABC-G4. Hence, cholesterol-conjugated iRNA agents could enjoy facilitated uptake by cells possessing such transport mechanisms, e.g. cells of the liver. As such, the present invention provides evidence and general methods for targeting iRNA agents to cells expressing certain cell surface components, e.g. receptors, by conjugating a natural ligand for such component (e.g. cholesterol) to the iRNA agent, or by conjugating a chemical moiety (e.g. cholesterol) to the iRNA agent which associates with or binds to a natural ligand for the component (e.g. LDL, HDL).

OTHER EMBODIMENTS

An iRNA agent, can be produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. Proc. Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of an iRNA agent and one that produces a transcript that includes the bottom strand of an iRNA agent. When the templates are transcribed, the iRNA agent is produced, and processed into siRNA agent fragments that mediate gene silencing.

Formulation

The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the dsRNAs of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl ethanolamine=DOPE, dimyristoylphosphatidyl choline=DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol=DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl=DOTAP and dioleoylphosphatidyl ethanolamine=DOTMA), e.g. (+/−)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide=GAP-DLRIE). DsRNAs of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which dsRNAs of the invention are administered in conjunction with one or more penetration enhancers, surfactants, and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories-surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell membrane, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidyl-ethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(m)1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(m)1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(m)1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_(1215G), that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁-C₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731) and other peptides, are also known to enhance the cellular uptake of dsRNAs.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Pharmaceutical Compositions for the Delivery to the Respiratory Tract

Another aspect of the invention provides for the delivery of iRNA agents to the respiratory tract, particularly for the treatment of cystic fibrosis. The respiratory tract includes the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conductive airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung. The epithelium of the conductive airways is the primary target of inhaled therapeutic aerosols for delivery of iRNA agents such as alpha-ENaC iRNA agents.

Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably the iRNA agent, within the dispersion can reach the lung where it can, for example, be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations; administration by inhalation may be oral and/or nasal. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. An iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

Examples of pharmaceutical devices for aerosol delivery include metered dose inhalers (MDIs), dry powder inhalers (DPIs), and air-jet nebulizers. Exemplary delivery systems by inhalation which can be readily adapted for delivery of the subject iRNA agents are described in, for example, U.S. Pat. Nos. 5,756,353; 5,858,784; and PCT applications WO98/31346; WO98/10796; WO00/27359; WO01/54664; WO02/060412. Other aerosol formulations that may be used for delivering the iRNA agents are described in U.S. Pat. Nos. 6,294,153; 6,344,194; 6,071,497, and PCT applications WO02/066078; WO02/053190; WO01/60420; WO00/66206. Further, methods for delivering iRNA agents can be adapted from those used in delivering other oligonucleotides (e.g., an antisense oligonucleotide) by inhalation, such as described in Templin et al., Antisense Nucleic Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al., Expert Opin Biol Ther, 2001, 1:979-83; Sandrasagra et al., Antisense Nucleic Acid Drug Dev, 2002, 12:177-81.

The delivery of the inventive agents may also involve the administration of so called “pro-drugs”, i.e. formulations or chemical modifications of a therapeutic substance that require some form of processing or transport by systems innate to the subject organism to release the therapeutic substance, preferably at the site where its action is desired; this latter embodiment may be used in conjunction with delivery of the respiratory tract, but also together with other embodiments of the present invention. For example, the human lungs can remove or rapidly degrade hydrolytically cleavable deposited aerosols over periods ranging from minutes to hours. In the upper airways, ciliated epithelia contribute to the “mucociliary excalator” by which particles are swept from the airways toward the mouth. Pavia, D., “Lung Mucociliary Clearance,” in Aerosols and the Lung: Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984. In the deep lungs, alveolar macrophages are capable of phagocytosing particles soon after their deposition. Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and Brain, J. D., “Physiology and Pathophysiology of Pulmonary Macrophages,” in The Reticuloendothelial System, S. M. Reichard and J. Filkins, Eds., Plenum, New. York., pp. 315-327, 1985.

In preferred embodiments, particularly where systemic dosing with the iRNA agent is desired, the aerosoled iRNA agents are formulated as microparticles. Microparticles having a diameter of between 0.5 and ten microns can penetrate the lungs, passing through most of the natural barriers. A diameter of less than ten microns is required to bypass the throat; a diameter of 0.5 microns or greater is required to avoid being exhaled.

Other Components

Compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical combinations and compositions containing (a) one or more dsRNA agents and (b) one or more other therapeutic agents which function by a non-RNA interference mechanism.

Accordingly, the invention includes a combination of an iRNA of the present invention with an anti-inflammatory, bronchodilatory, antihistamine, anti-tussive, antibiotic or DNase drug substance, said epithelial sodium channel blocker and said drug substance being in the same or different pharmaceutical composition.

Suitable antibiotics include macrolide antibiotics, e.g., tobramycin (TOBI™).

Suitable DNase drug substances include dornase alfa (Pulmozyme™), a highly-purified solution of recombinant human deoxyribonuclease I (rhDNase), which selectively cleaves DNA. Dornase alfa is used to treat cystic fibrosis.

Other useful combinations of epithelial sodium channel blockers with anti-inflammatory drugs are those with antagonists of chemokine receptors, e.g., CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5 antagonists, such as Schering-Plough antagonists SC-351125, SCH-55700 and SCH-D; Takeda antagonists, such as N-[[4-[[[6,7-dihydro-2-(4-methyl-phenyl)-5H-benzo-cyclohepten-8-yl]carbonyl]amino]phenyl]-methyl]tetrahydro-N,N-dimethyl-2H-pyran-4-amin-ium chloride (TAK-770); and CCR-5 antagonists described in U.S. Pat. No. 6,166,037 (particularly claims 18 and 19), WO 00/66558 (particularly claim 8), WO 00/66559 (particularly claim 9), WO 04/018425 and WO 04/026873.

Suitable anti-inflammatory drugs include steroids, in particular, glucocorticosteroids, such as budesonide, beclamethasone dipropionate, fluticasone propionate, ciclesonide or mometasone furoate, or steroids described in WO 02/88167, WO 02/12266, WO 02/100879, WO 02/00679 (especially those of Examples 3, 11, 14, 17, 19, 26, 34, 37, 39, 51, 60, 67, 72, 73, 90, 99 and 101), WO 03/35668, WO 03/48181, WO 03/62259, WO 03/64445, WO 03/72592, WO 04/39827 and WO 04/66920; non-steroidal glucocorticoid receptor agonists, such as those described in DE 10261874, WO 00/00531, WO 02/10143, WO 03/82280, WO 03/82787, WO 03/86294, WO 03/104195, WO 03/101932, WO 04/05229, WO 04/18429, WO 04/19935 and WO 04/26248; LTD4 antagonists, such as montelukast and zafirlukast; PDE4 inhibitors, such as cilomilast (Ariflo® GlaxoSmithKline), Roflumilast (Byk Gulden), V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-Plough), Arofylline (Almirall Prodesfarma), PD189659/PD168787 (Parke-Davis), AWD-12-281 (Asta Medica), CDC-801 (Celgene), SelCID(TM) CC-10004 (Celgene), VM554/UM565 (Vernalis), T-440 (Tanabe), KW-4490 (Kyowa Hakko Kogyo), and those disclosed in WO 92/19594, WO 93/19749, WO 93/19750, WO 93/19751, WO 98/18796, WO 99/16766, WO 01/13953, WO 03/104204, WO 03/104205, WO 03/39544, WO 04/000814, WO 04/000839, WO 04/005258, WO 04/018450, WO 04/018451, WO 04/018457, WO 04/018465, WO 04/018431, WO 04/018449, WO 04/018450, WO 04/018451, WO 04/018457, WO 04/018465, WO 04/019944, WO 04/019945, WO 04/045607 and WO 04/037805; adenosine A2B receptor antagonists such as those described in WO 02/42298; and beta-2 adrenoceptor agonists, such as albuterol (salbutamol), metaproterenol, terbutaline, salmeterol fenoterol, procaterol, and especially, formoterol, carmoterol and pharmaceutically acceptable salts thereof, and compounds (in free or salt or solvate form) of formula (I) of WO 0075114, which document is incorporated herein by reference, preferably compounds of the Examples thereof, especially indacaterol and pharmaceutically acceptable salts thereof, as well as compounds (in free or salt or solvate form) of formula (I) of WO 04/16601, and also compounds of EP 1440966, JP 05025045, WO 93/18007, WO 99/64035, USP 2002/0055651, WO 01/42193, WO 01/83462, WO 02/66422, WO 02/70490, WO 02/76933, WO 03/24439, WO 03/42160, WO 03/42164, WO 03/72539, WO 03/91204, WO 03/99764, WO 04/16578, WO 04/22547, WO 04/32921, WO 04/33412, WO 04/37768, WO 04/37773, WO 04/37807, WO 04/39762, WO 04/39766, WO 04/45618, WO 04/46083, WO 04/80964, WO 04/108765 and WO 04/108676.

Suitable bronchodilatory drugs include anticholinergic or antimuscarinic agents, in particular, ipratropium bromide, oxitropium bromide, tiotropium salts and CHF 4226 (Chiesi), and glycopyrrolate, but also those described in EP 424021, U.S. Pat. Nos. 3,714,357, 5,171,744, WO 01/04118, WO 02/00652, WO 02/51841, WO 02/53564, WO 03/00840, WO 03/33495, WO 03/53966, WO 03/87094, WO 04/018422 and WO 04/05285.

Suitable dual anti-inflammatory and bronchodilatory drugs include dual beta-2 adrenoceptor agonist/muscarinic antagonists such as those disclosed in USP 2004/0167167, WO 04/74246 and WO 04/74812.

Suitable antihistamine drug substances include cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratidine, desloratidine, diphenhydramine and fexofenadine hydrochloride, activastine, astemizole, azelastine, ebastine, epinastine, mizolastine and tefenadine, as well as those disclosed in JP 2004107299, WO 03/099807 and WO 04/026841.

Other useful combinations of agents of the invention with anti-inflammatory drugs are those with antagonists of chemokine receptors, e.g., CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5 antagonists, such as Schering-Plough antagonists SC-351125, SCH-55700 and SCH-D; Takeda antagonists, such as N-[[4-[[[6,7-dihydro-2-(4-methylphenyl)-5H-benzo-cyclohepten-8-yl]carbonyl]amino]phenyl]-methyl]tetrahydro-N,N-dimethyl-2H-pyran-4-amin-ium chloride (TAK-770), and CCR-5 antagonists described in U.S. Pat. No. 6,166,037 (particularly claims 18 and 19), WO 00/66558 (particularly claim 8), WO 00/66559 (particularly claim 9), WO 04/018425 and WO 04/026873.

Other useful additional therapeutic agents may also be selected from the group consisting of cytokine binding molecules, particularly antibodies of other cytokines, in particular a combination with an anti-IL4 antibody, such as described in PCT/EP2005/00836, an anti-IgE antibody, such as Xolair®, an anti-IL31 antibody, an anti-IL31R antibody, an anti-TSLP antibody, an anti-TSLP receptor antibody, an anti-endoglin antibody, an anti-IL1b antibody or an anti-IL13 antibody, such as described in WO05/007699.

Two or more combined compounds may be used together in a single formulation, separately, concomitantly or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions of the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of ENaC related disorders. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Treatment Methods and Routes of Delivery

A composition that includes an iRNA agent, e.g., an iRNA agent that targets alpha-ENaC, can be delivered to a subject by a variety of routes to achieve either local delivery to the site of action or systemic delivery to the subject. Exemplary routes include direct local administration to the site of treatment, such as the lungs and nasal passage as well as intravenous, nasal, oral, and ocular delivery. The preferred means of administering the iRNA agents of the present invention is through direct administration to the lungs and nasal passage as a liquid, aerosol or nebulized solution.

Formulations for inhalation or parenteral administration are well known in the art. Such formulation may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

The active compounds disclosed herein are preferably administered to the lung(s) or nasal passage of a subject by any suitable means. Active compounds may be administered by administering an aerosol suspension of respirable particles comprised of the active compound or active compounds, which the subject inhales. The active compound can be aerosolized in a variety of forms, such as, but not limited to, dry powder inhalants, metered dose inhalants, or liquid/liquid suspensions. The respirable particles may be liquid or solid. The particles may optionally contain other therapeutic ingredients such as amiloride, benzamil or phenamil, with the selected compound included in an amount effective to inhibit the reabsorption of water from airway mucous secretions, as described in U.S. Pat. No. 4,501,729.

The particulate pharmaceutical composition may optionally be combined with a carrier to aid in dispersion or transport. A suitable carrier such as a sugar (i.e., lactose, sucrose, trehalose, mannitol) may be blended with the active compound or compounds in any suitable ratio (e.g., a 1 to 1 ratio by weight).

Particles comprised of the active compound for practicing the present invention should include particles of respirable size, that is, particles of a size sufficiently small to pass through the mouth or nose and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 10 microns in size (more particularly, less than about 5 microns in size) are respirable. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. For nasal administration, a particle size in the range of 10-500 uM is preferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing an aerosol may be prepared by combining the active compound with a suitable vehicle, such as sterile pyrogen free water. The hypertonic saline solutions used to carry out the present invention are preferably sterile, pyrogen-free solutions, comprising from one to fifteen percent (by weight) of the physiologically acceptable salt, and more preferably from three to seven percent by weight of the physiologically acceptable salt.

Aerosols of liquid particles comprising the active compound may be produced by any suitable means, such as with a pressure-driven jet nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.

Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w/w of the formulation, but preferably less than 20% w/w. The carrier is typically water (and most preferably sterile, pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic, but may be hypertonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate therapeutic aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable and generate a volume of aerosol containing a predetermined metered dose of a therapeutic at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation.

A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquefied propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 200 ul, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidant and suitable flavoring agents.

An iRNA agent can be incorporated into pharmaceutical compositions suitable for administration. For example, compositions can include one or more species of an iRNA agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Administration can be provided by the subject or by another person, e.g., a caregiver. A caregiver can be any entity involved with providing care to the human: for example, a hospital, hospice, doctor's office, outpatient clinic; a healthcare worker such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent. The medication can be provided in measured doses or in a dispenser which delivers a metered dose.

The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.

The term “co-administration” refers to administering to a subject two or more agents, and in particular two or more iRNA agents. The agents can be contained in a single pharmaceutical composition and be administered at the same time, or the agents can be contained in separate formulation and administered serially to a subject. So long as the two agents can be detected in the subject at the same time, the two agents are said to be coadministered.

The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

Dosage

An iRNA agent can be administered at a unit dose less than about 75 mg per kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of bodyweight, and less than 200 nmol of iRNA agent (e.g., about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmol of iRNA agent per kg of bodyweight. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular, intrathecally, or directly into an organ), an inhaled dose, or a topical application.

The dosage can be an amount effective to treat or prevent a disease or disorder. It can be given prophylactically or as the primary or a part of a treatment protocol.

In one embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time. Because iRNA agent mediated silencing can persist for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen.

In one embodiment, a subject is administered an initial dose, and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into an siRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 to 75 mg/kg of body weight per day, e.g., 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of body weight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.001 g to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, or pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage of an iRNA agent such as an siRNA used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays. For example, the subject can be monitored after administering an iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models as described above.

iRNA agents of the present invention as described herein may be useful in the treatment and (where appropriate) in the prevention of any one of the following diseases/disorders;

Cystic fibrosis, Liddles syndrome, renal insufficiency, hypertension, electrolyte imbalances.

In particular in some embodiments, iRNA agents of the invention may be used to treat and/or prevent adverse clinical manifestations of these diseases/disorders.

The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES

Source of reagents Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Example 1: Selection of Sequences

In order to identify therapeutic siRNAs to downmodulate expression of the alpha subunit of the epithelial sodium channel ENaC (α-ENaC), screening sets were defined based on a bioinformatic analysis. The key drivers for the design of the screening set were predicted specificity of the siRNAs against the transcriptome of the relevant species. For the identification of alpha-ENaC siRNAs and an efficient delivery system a three pronged approach was used: Rat was selected as the test species to address silencing efficacy in vivo after intratracheal delivery, guinea pig was selected as the disease model organism to demonstrate that alpha-ENaC mRNA reduction results in a measurable functional effect. The therapeutic siRNA molecule has to target human alpha-ENaC as well as the alpha-ENaC sequence of at least one toxicology-relevant species, in this case, rhesus monkey.

Initial analysis of the relevant alpha-ENaC mRNA sequence revealed few sequences can be identified that fulfil the specificity requirements and at the same time target alpha-ENaC mRNA in all relevant species. Therefore it was decided to design independent screening sets for the therapeutic siRNA and for the surrogate molecules to be tested in the relevant disease model (Tables 1A, 1B, 1C and 1D).

All siRNAs recognize the human alpha-ENaC sequence, as a human cell culture system was selected for determination of in vitro activity (H441, see below). Therefore all siRNAs can be used to target human alpha-ENaC mRNA in a therapeutic setting.

The therapeutic screening sets were designed to contain only siRNA sequences that are fully complementary to the human and rhesus monkey alpha-ENaC sequences.

Design and in silico selection of siRNAs targeting alpha-ENaC (SCNN1A) siRNA design was carried out to identify siRNAs for the four previously defined sets (see above)

a) “Initial screening set”

b) “Extended screening set”

c) “In vivo surrogate set for rat”

d) “In vivo surrogate set for guinea pig”

Initial screening set The aim for in silico selection of an initial screening set was to identify siRNAs specifically targeting human alpha-ENaC, as well as its rhesus monkey ortholog. The human target mRNA (NM_001038.4) was downloaded from NCBI resource (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide)_during the complete siRNA selection procedure. In order to identify the alpha-ENaC rhesus (Macaca mulatta) ortholog, the human sequence was used in a blastn search at Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu/blast/?organism=Mmulatta) against Mmulatta contigs as of 2004 10 01. All hit regions were extracted and assembled by the CAP assembly tool to generate a first assembly sequence. Further, a BLAST search was performed with the human sequence at UCSC (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start&org=Rhesus&db=rheMac2&hgsid=84859356) against Rhesus freeze 12 Mar. 2005. The scaffold hit 84554 was downloaded and used together with the first assembly sequence by CAP to generate the final consensus sequence for rhesus alpha-ENaC.

Following extraction of all overlapping 19mer sequences out of the human mRNA, conserved 19mers were identified that had identical sequences in the assembled rhesus consensus sequence. Those 19mer sequences were defined as the pool of human-rhesus cross-reactive siRNA (sense) sequences, represented by 1185 19mers.

The corresponding antisense sequences were generated and tested for specificity in human. For this, their predicted potential for interacting with irrelevant target mRNAs (off-target potential) was taken as parameter. Sequences with low off-target potential were defined as preferable and predicted to be more specific.

For further selection, candidate siRNAs were ranked according to their predicted potential for interacting with other host sequences (here, without limitation, human). siRNAs with low off-target potential are assumed to be more specific in vivo. For predicting siRNA-specific off-target potential, the following assumptions were made:

-   -   1) off-target potential of a strand can be deduced from the         number and distribution of mismatches to an off-target     -   2) the most relevant off-target, that is the gene predicted to         have the highest probability to be silenced due to tolerance of         mismatches, determines the off-target potential of the strand     -   3) positions 2 to 9 (counting 5′ to 3′) of a strand (seed         region) may contribute more to off-target potential than rest of         sequence (that is non-seed and cleavage site region) (Haley, B.,         and Zamore, P. D., Nat Struct Mol Biol. 2004, 11:599).     -   4) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage         site region) may contribute more to off-target potential than         non-seed region (that is positions 12 to 18, counting 5′ to 3′)     -   5) positions 1 and 19 of each strand are not relevant for         off-target interactions     -   6) off-target potential can be expressed by the off-target score         of the most relevant off-target, calculated based on number and         position of mismatches of the strand to the most homologous         region in the off-target gene considering assumptions 3 to 5     -   7) assuming potential abortion of sense strand activity by         internal modifications introduced, only off-target potential of         antisense strand will be relevant

To identify potential off-target genes, 19mer antisense sequences were subjected to a homology search against publicly available human mRNA sequences, assumed to represent the human comprehensive transcriptome.

To this purpose, fastA (version 3.4) searches were performed with all 19mer sequences against a human RefSeq database (available version from ftp://ftp.ncbi.nih.gov/refseq/ on Nov., 18 2005). FastA search was executed with parameters-values-pairs-f 30-g 30 in order to take into account the homology over the full length of the 19mer without any gaps. In addition, in order to ensure the listing of all relevant off-target hits in the fastA output file the parameter-E 15000 was used.

The search resulted in a list of potential off-targets for each input sequence listed by descending sequence homology over the complete 19mer.

To rank all potential off-targets according to assumptions 3 to 5, and by this identify the most relevant off-target gene and its off-target score, fastA output files were analyzed by a perl script.

The script extracted the following off-target properties for each 19mer input sequence and each off-target gene to calculate the off-target score:

-   -   Number of mismatches in non-seed region     -   Number of mismatches in seed region     -   Number of mismatches in cleavage site region         The off-target score was calculated by considering assumptions 3         to 5 as follows:

$\begin{matrix} {{{Off–target}\mspace{14mu}{score}} = {{number}\mspace{14mu}{of}\mspace{14mu}{seed}\mspace{14mu}{mismatches}*10}} \\ {= {{number}\mspace{14mu}{of}\mspace{14mu}{cleavage}\mspace{14mu}{site}\mspace{14mu}{mismatches}*1.2}} \\ {= {{number}\mspace{14mu}{of}\mspace{14mu}{{non}–{seed}}\mspace{14mu}{mismatches}*1}} \end{matrix}$

The most relevant off-target gene for each 19mer sequence was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as representative for the off-target potential of each siRNA, represented by the 19mer antisense sequence analyzed.

Calculated off-target potential was used as sorting parameter (descending by off-target score) in order to generate a ranking for all human-rhesus cross-reactive siRNA sequences.

An off-target score of 3 or more was defined as prerequisite for siRNA selection, whereas all sequences containing 4 or more G's in a row (poly-G sequences) were excluded, leading to selection of a total of 152 siRNAs targeting human and rhesus ENaC alpha (see Table 1a).

Extended screening set The aim for in silico selection of the extended screening set was to identify all further siRNAs targeting human alpha-ENaC with sufficient specificity, that were excluded from the initial set due to missing cross-reactivity to rhesus. The remaining sequences from the pool of 19mers derived from human alpha-ENaC that have not been analyzed before were taken and the corresponding antisense sequences were generated. The most relevant off-target gene and its corresponding off-target scores were calculated as described in section “Initial screening set”.

For determining cross-reactivity to mouse and guinea pig (Cavia porcellus/cobya), alpha-ENaC sequences of these species were downloaded from NCBI nucleotide database¹ (accession numbers NM_011324.1 and AF071230 (full length)/DQ109811 (partial cds), respectively). The two guinea pig sequences were used to generate aguinea pig alpha-ENaC consensus sequence. Every human 19mer sequence was tested for presence in the mouse and guinea pig sequences. Positive sequences were assigned to the pool of human-mouse cross-reactive siRNA (sense) sequences, or human-guinea pig cross-reactive siRNA (sense) sequences. After exclusion of all poly-G sequences, sequences were selected with off-target scores of 3 or more as well as those with off-target scores of 2.2 or 2.4 and cross-reactivity to mouse, rhesus or guinea pig. The total number of siRNAs in the extended screening pool was 344 (see Table 1b).

In vivo rat surrogate set The aim for in silico selection of the in vivo rat surrogate set was to identify all siRNAs targeting human and rat alpha-ENaC with sufficient specificity in rat. For identification of human-rat cross-reactive siRNAs, rat alpha-ENaC mRNA sequence was downloaded from NCBI nucleotide database (accession number, NM_031548.2), and all sequences out of the pool of human 19mers were tested for presence in the rat sequence, representing the pool of human-rat cross-reactive siRNA (sense) sequences.

The corresponding antisense sequences were generated and tested for specificity in rat. For this, the most relevant off-target gene in rat and its corresponding off-target scores were calculated as described in section “Initial screening set” using the rat mRNA set (RefSeq database) instead of the human transcripts. After exclusion of all poly-G sequences, a ranking was generated considering the rat off-target score in first priority and the human off-target score with second priority. Those 48 sequences from the top of the list were finally selected representing the in vivo rat surrogate set (see Table 1c).

In vivo guinea pig surrogate set The aim for in silico selection of the in vivo guinea pig surrogate set was to identify all siRNAs targeting human and guinea pig alpha-ENaC that have not been selected in previous sets. The remaining siRNAs of the previously determined set of human-guinea pig cross-reactive siRNA (sense) sequences were ranked according to human off-target scores. The top 63 sequences (excluding poly-G sequences) were selected, representing the in vivo guinea pig surrogate set (see Table 1d).

Example 2: siRNA Synthesis

Synthesis of nucleotides comprising natural bases. As the siRNAs from the screening sets are all potentially intended for in vivo administration, siRNAs were synthesised with a modification strategy that protects the siRNAs from degradation by endo- and exonucleases in a biological environment. In this strategy, the 3′-ends of both strands are protected from a 3′->5′-exonucleotitic activity by a phosphorothioate linkage between the two last nucleobases at the 3′-end. In order to inhibit endo-nucleolytic degradation of the siRNA all pyrimidines in the sense strand of the siRNA were replaced with the corresponding 2′-O-methyl-modified ribonucleotide. To reduce the number of modifications in the antisense strand, which is the more active strand and therefore more sensitive to modifications, we only modified the pyrimidines in the context of previously identified major nuclease cleavage sites with 2′-O-methyl groups. The major cleavage sites are the following two sequence motifs: 5′-UA-3′ and 5′-CA-3′.

Since it has also been considered to use siRNAs in formulations that potentially protect the RNAs from the nucleolytic biological environment in the lung, the same set of siRNAs were also synthesized without any protection from endonucleolytic degradation.

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleißheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was diluted to a concentration of 50 μmole double stranded RNA/L and stored at −20° C. until use.

Example 3: siRNA Testing In Vitro

The ability of the iRNA agents to inhibit expression of alpha-ENaC was tested in human cell lines in vitro, or in rats in vivo. The iRNA agent is transfected into the cells, e.g., by transfection, allowed to act on the cells for a certain time, e.g., 24 hours, and levels of alpha-ENaC mRNA were determined by branched-DNA analysis. Alternatively, the iRNA agent is administered in vivo via the intratracheal route and the inhibition of alpha-ENaC mRNA expression determined by branched-DNA analysis on the target organ. Complementing these direct assays, we tested the inhibition of target gene expression by RNAi agents for alpha-ENaC mRNA recombinantly expressed in mammalian host cells.

Cell lines. H441 cells were obtained from the American Type Culture Collection (ATCC-Number: HTB-174, LCG Promochem GmbH, Wesel, Germany) and were grown in RPMI 1640, 10% fetal calf serum, 100u penicillin/100 μg/mL streptomycin, 2 mM L-glutamine, 10 nM Hepes and 1 mM Sodium-Pyruvate (all from Biochrom AG, Berlin, Germany) at 37° C. under a 5% CO₂/95% air atmosphere.

Primary human bronchial epithelial cells were obtained from Cambrex (Cat #CC-2540) and were routinely grown in BEGM media with singlequots (Cambrex Cat #CC-3170 minus tri-iodothreonine). For polarisation and growth at air liquid interface the cells were grown in a 1:1 mixture of BEGM:DMEM supplemented with 4.5 g/L D-Glucose (Gibco BRL Cat #41965-039) and supplemented with singlequots (Cambrex Cat #CC-4175), as above but minus the tri-iodothreonine and GA1000 aliquots and in the presence of 50 g/mL Gentamycin (Gibco Brl Cat #10131-015). As cells were maintained in serum-free media, trypsin neutralisation solution was used during passaging steps (Cambrex Cat #CC-5002). For polarisation and culture at air-liquid interface the cells were grown on semipermeable (0.4 micron) polycarbonate supports (Corning Costar Cat #3407 #3460) and cultured throughout at 37° C. under a 5% CO₂/95% air atmosphere.

Cos-1 African green monkey kidney cells (ATCC #CRL-1650) were grown in Dulbecco's MEM, 4.5 g/L glucose, 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate (Gibco BRL), 100u penicillin/100 μg/mL streptomycin.

Example 3.1: In Vitro Screen for Active Alpha-ENaC siRNAs and IC50 Determination in H441

One day prior to transfection, ENaC-alpha expression was induced in H441 cells (ATCC-Number: HTB-174, LCG Promochem GmbH, Wesel, Germany) by adding 100 nM of dexamethasone. Directly before transfection, cells were seeded at 1.5×10⁴ cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μL of growth medium (RPMI 1640, 10% fetal calf serum, 100u penicillin/100 μg/ml streptomycin, 2 mM L-glutamine, 10 nM Hepes and 1 mM Sodium-Pyruvate, all from Biochrom AG, Berlin, Germany). Transfections were performed in quadruplicates. For each well 0.5 μL Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μL Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. For the siRNA concentration being 50 nM in the 100 μL transfection volume, 1 μL of a 5 μM siRNA were mixed with 11.5 μL Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. siRNA-Lipofectamine2000-complexes were applied completely (25 μL each per well) to the cells and cells were incubated for 24 h at 37° C. and 5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau).

Cells were harvested by applying 50 μL of lysis mixture (content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well containing 100 μL of growth medium and were lysed at 53° C. for 30 min. Afterwards, 50 μL of the lysates were incubated with probesets specific to human ENaC-alpha and human GAPDH (sequence of probesets see below) and proceeded according to the manufacturer's protocol for QuantiGene. In the end chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the hENaC probeset were normalized to the respective GAPDH values for each well. Values obtained with siRNAs directed against ENaC-alpha were related to the value obtained with an unspecific siRNA (directed against HCV) which was set to 100%. The percentage residual expression of alpha-ENaC for siRNA examples is shown in Tables 1A-1D.

Effective siRNAs from the screen were further characterized by dose response curves. Transfections of dose response curves were performed at the following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77 picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9 fM and mock (no siRNA) and diluted with Opti-MEM to a final concentration of 12.5 μl according to the above protocol. Data analysis was performed using Microsoft Excel add-in software XL-fit 4.2 (IDBS, Guildford, Surrey, UK) and applying the sigmoidal model number 606.

Probesets:

SEQ. ID. FPL Name Members Function Sequence NO: human alpha-ENaC: hENAC001 .235.255.CE CE gtctgtccagggtttccttccTTTTTctcttggaaagaaagt 1645 hENAC002 .274.293.CE CE actgccattcttggtgcagtTTTTTctcttggaaagaaagt 1646 hENAC003 .344.367.CE CE ctctcctggaagcaggagtgaataTTTTTctcttggaaagaaagt 1647 hENAC004 .391.411.CE CE gccgcggatagaagatgtaggTTTTTctcttggaaagaaagt 1648 hENAC005 .501.521.CE CE gcacttggtgaaacagcccagTTTTTctcttggaaagaaagt 1649 hENAC006 .539.560.CE CE agcagagagctggtagctggtcTTTTTctcttggaaagaaagt 1650 hENAC007 .256.273.LE LE cgccataatcgcccccaaTTTTTaggcataggacccgtgtct 1651 hENAC008 .368.390.LE LE cacagccacactccttgatcatgTTTTTaggcataggacccgtgtct 1652 hENAC009 .412.431.LE LE acagtactccacgttctgggTTTTTaggcataggacccgtgtct 1653 hENAC010 .455.477.LE LE ggagcttatagtagcagtaccccTTTTTaggcataggacccgtgtct 1654 hENAC011 .522.538.LE LE acgctgcatggcttccgTTTTTaggcataggacccgtgtct 1655 hENAC012 .561.580.LE LE gagggccatcgtgagtaaccTTTTTaggcataggacccgtgtct 1656 hENAC013 .214.234.BL BL Tcatgctgatggaggtctcca 1657 hENAC014 .294.318.BL BL Ggtaaaggttctcaacaggaacatc 1658 hENAC015 .319.343.BL BL Cacacctgctgtgtgtactttgaag 1659 hENAC016 .432.454.BL BL Caggaactgtgctttctgtagtc 1660 hENAC017 .478.500.BL BL Gtggtctgaggagaagtcaacct 1661 hENAC018 .581.599.BL BL Ccattcctgggatgtcacc 1662 human GAPDH: hGAP001 AF261085.252.271.CE CE gaatttgccatgggtggaatTTTTTctcttggaaagaaagt 1663 hGAP002 AF261085.333.352.CE CE ggagggatctcgctcctggaTTTTTctcttggaaagaaagt 1664 hGAP003 AF261085.413.431.CE CE ccccagccttctccatggtTTTTTctcttggaaagaaagt 1665 hGAP004 AF261085.432.450.CE CE gctcccccctgcaaatgagTTTTTctcttggaaagaaagt 1666 hGAP005 AF261085.272.289.LE LE agccttgacggtgccatgTTTTTaggcataggacccgtgtc 1667 t hGAP006 AF261085.290.310.LE LE gatgacaagcttcccgttctcTTTTTaggcataggacccgt 1668 gtct hGAP007 AF261085.311.332.LE LE agatggtgatgggatttccattTTTTTaggcataggacccgt 1669 gtct hGAP008 AF261085.353.372.LE LE gcatcgccccacttgattttTTTTTaggcataggacccgtgt 1670 ct hGAP009 AF261085.373.391.LE LE cacgacgtactcagcgccaTTTTTaggcataggacccgt 1671 gtct hGAP010 AF261085.451.472.LE LE ggcagagatgatgacccttttgTTTTTaggcataggaccc 1672 gtgtct hGAP011 AF261085.392.412.BL BL Ggtgaagacgccagtggactc 1673

The IC₅₀s for siRNA examples is shown in Table 2A and 2B.

Example 3.2: Transient Alpha-ENaC Knockdown in a Primary Human Bronchial Epithelial Model

Human bronchial epithelial cells (donor reference 4F1499) were plated in 24-well plates at 1×10⁵ cells per well in 0.5 mL growth medium one day before transfection. The cells were 70% confluent on the day of siRNA transfection.

Each siRNA was resuspended at 100 nM in 1 mL of Optimem I (Invitrogen) and in a separate tube, Lipofectamine 2000 (Invitrogen) was diluted to 6 L/mL in Optimem, giving an amount sufficient for transfection of four replicates in a 24-well plate. After 5 minutes at room temperature, the mixtures were combined to give the desired final concentration of 50 nM siRNA and 3 L/mL Lipofectamine 2000. The transfection mixture was incubated for a further 20 minutes at room temperature and 420 μL of the siRNA/reagent complex was added to each well as dictated by the experimental design. Plates were gently rocked to ensure complete mixing and then incubated at 37° C. in an incubator at 5% CO₂/95% air for 4 hours. Subsequently, the transfection mixture was aspirated and the cells were returned to normal culture conditions for a further 20 hours.

Cell lysates were prepared for branched-DNA analysis. A 2:1 medium:lysis buffer (Panomics) mixture was prepared and cells were lysed in 200 μL at 53° C. for 30 minutes. After a visual check for complete lysis, the cell lysates were stored at −80° C. for subsequent analysis. Branched-DNA analysis was performed as described above, with alpha-ENaC expression normalized against GAPDH. The branched DNA analysis protocol used differs from that above only in that 20 μL of sample was applied to each well in this case.

Table 2C shows the alpha-ENaC expression in primary HBEC for siRNA examples.

Example 3.3: In Vitro Inhibition of Exogenously Expressed Cloned Cynomolgous Alpha-ENaC Gene Expression for Selected RNAi Agents in Cos-1 Cells

Cloning of the cynomolgous alpha-ENaC sequence.

Primer sequences for amplification of 5′- UTR and CDS (nucleotides shown in brackets correspond to the Macaca mulatta (Rhesus monkey) α-ENaC cDNA sequence): P745: (SEQ. I.D. NO: 1674) 5′ - CTCCATGTTCTGCGGCCGCGGATAGAAG-3′ (nt 1427) P733: (SEQ. I.D. NO: 1675) 5′- CCGGCCGGCGGGCGGGCT-3′ (nt 1) P734: (SEQ. I.D. NO: 1676) 5- CTCCCCAGCCCGGCCGCT-3′ (nt 17) P735: (SEQ. I.D. NO: 1677) 5′- GGCCGCTGCACCTGTAGGG-3′ (nt 28) Primer sequences for amplification of CDS and 3′- UTR: P737: (SEQ. I.D. NO: 1678) 5′- ATGGAGTACTGTGACTACAGG-3′ (nt 1422) P740: (SEQ. I.D. NO: 1679) 5′- TTGAGCATCTGCCTACTTG-3′ (nt 3113) Primer sequences for amplification of internal part of CDS: P713: (SEQ. I.D. NO: 1679) 5′- 5′-ATGGATGATGGTGGCTTTAACTTGCGG-3′ (nt 1182) P715: (SEQ. I.D. NO: 1680) 5′- 5′-TCAGGGCCCCCCCAGAGG-3′ (nt 2108)

Cynomolgus (Macaca fascicularis) lung total RNA (#R1534152-Cy-BC) was purchased from BioCat (Germany). Synthesis of cDNA was performed using the SuperScript III First Strand Synthesis System (Invitrogen). Synthesis of cDNA was performed using either random hexamers or oligo dT primers. In addition, cynomolgus lung first strand cDNA was also purchased from BioCat/#C1534160-Cy-BC). For PCR amplification, the Advantage 2 PCR kit (#K1910-1, Clontech) was used. Amplification of the 5′-UTR and parts of the CDS was performed using P745 and a equimolar mixture of P733, P734 and P735. For PCR amplification of the CDS and 3′-UTR, primers P737 and P740 were used. The primers P713 and P715 were used for amplification of parts of the CDS.

All PCR products were analysed by agarose gel electrophoresis and then cloned into the pCR2.1 vector using the TOPO TA cloning kit (Invitrogen) in TOP10 bacteria. Clones were then picked and DNA was isolated using the Qiagen Miniprep kit. After restriction enzyme digest with EcoRI and analysis by agarose gel electrophoresis, DNA from correct clones were subjected to sequencing.

The sequences were then aligned with the α-ENaC cDNA sequence of Rhesus monkey, and sequences of the individual clones were aligned with each other. The full-length cynomolgus alpha-ENaC cDNA was then cloned by digestion of the 5′-part (5′-UTR and CDS, clone 55) with EcoR I and Not I, digestion of the middle part of the CDS by Not I and BstE II (clone 15), and the 3′-part (CDS and 3′-UTR) by BstEII and EcoR V (clone 80). The digested DNA fragments were then subcloned into pcDNA3.1, digested with EcoR I and EcoR V. The full-length cynomolgus alpha-ENaC cDNA in pcDNA3.1 was then subjected to full-length sequencing (Ingenetix, Vienna, Austria). The cynomolgus alpha-ENaC cDNA sequence corresponds to nt 28-3113 of the Rhesus alpha-ENaC cDNA sequence. Finally the cynomolgus alpha-ENaC cDNA was then excised from pcDNA3.1-cynomolgus alpha-ENaC by digestion with BamH I and EcoR V and subcloned into the vector pXOON. The plasmid map is illustrated in FIG. 1. FIG. 2 depicts the protein (SEQ.I.D.NO:1681) and DNA (SEQ.I.D.NO:1682) sequence of cynomolgous alpha-ENaC.

Transfections: COS-1 cells were seeded at 6×10⁴ cells/well on 24 well plates each in 0.5 mL of growth medium. One day after seeding the cells were co-transfected with the pXOON cynomolgous alpha-ENaC expression plasmid and the indicated siRNA. For each well, 4 ng of alpha-ENaC expression plasmid and 600 ng carrier plasmid (pNFAT-luc) were co-transfected with the relevant siRNA (final concentration 45 nM) using X-treme gene transfection reagent (Roche) at 3.75 μL/well in a total volume of 720 L/well Opti-MEM (Invitrogen) as described below.

Transfections were performed in triplicate for each sample. Plasmid/siRNA mastermixes (each for 3.5 wells) were prepared as follows: 14 ng alpha-ENaC expression plasmid, 2.1 μg carrier plasmid and 112 μmoles of relevant siRNA in a total volume 210 μL (Opti-MEM). A lipid mastermix was prepared for the whole transfection (105 μL lipid plus 1575 μL Opti-MEM for eight triplicate transfection samples). Plasmid/siRNA and lipid were mixed in equal volume to give a total volume of 420 μL transfection mix per triplicate sample (3.5×). Following a 20 minute incubation at room temperature, 120 μL of the relevant transfection mix was added to each well of cells in a final transfection volume of 720 μL (Opti-MEM). Cells were transfected for 24 hours at 37° C. and 5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau, Germany) and harvested for branched-DNA analysis.

Cell lysates were prepared for branched DNA analysis. A 2:1 medium:lysis buffer (Panomics) mixture was prepared and cells were lysed in 200 μL at 53° C. for 30 minutes. After a visual check for complete lysis, the cell lysates were stored at −80° C. for subsequent analysis. Branched-DNA analysis was performed as described above, with cyno alpha-ENaC expression normalized against eGFP from the expression plasmid. The branched-DNA analysis protocol used differs from that above only in that 20 μL of sample was applied to each well in this case.

FPL Name Function Sequence SEQ ID NO: Probesets: cynomolgous alpha-ENaC: cyENa001 CE cgccgtgggctgctgggTTTTTctcttggaaagaaagt 1683 cyENa002 CE ggtaggagcggtggaactcTTTTTctcttggaaagaaagt 1684 cyENa003 CE cagaagaactcgaagagctctcTTTTTctcttggaaagaaagt 1685 cyENa004 CE cccagaaggccgtcttcatTTTTTctcttggaaagaaagt 1686 cyENa005 LE ggtgcagagccagagcactgTTTTTctcttggaaagaaagt 1687 cyENa006 LE gtgccgcaggttctgggTTTTTaggcataggacccgtgtct 1688 cyENa007 LE gatcagggcctcctcctcTTTTTaggcataggacccgtgtct 1689 cyENa008 LE ccgtggatggtggtattgttgTTTTTaggcataggacccgtgtct 1690 cyENa009 LE gcggttgtgctgggagcTTTTTaggcataggacccgtgtct 1691 cyENa0010 LE ttgccagtacatcatgccaaaTTTTTaggcataggacccgtgtct 1692 cyENa0011 BL acaccaggcggatggcg 1693 eGFP: EGFP001 CE ggcacgggcagcttgcTTTTTctcttggaaagaaagt 1694 EGFP002 CE ggtagcggctgaagcactgTTTTTctcttggaaagaaagt 1695 EGFP003 CE cctggacgtagccttcgggTTTTTctcttggaaagaaagt 1696 EGFP004 CE ccttgaagaagatggtgcgctTTTTTctcttggaaagaaagt 1697 EGFP005 LE cgaacttcacctcggcgcTTTTTctcttggaaagaaagt 1698 EGFP006 LE ccttcagctcgatgcggtTTTTTctcttggaaagaaagt 1699 EGFP007 LE gtcacgagggtgggccagTTTTTaggcataggacccgtgtct 1700 EGFP008 LE cacgccgtaggtcagggtgTTTTTaggcataggacccgtgtct 1701 EGFP009 LE gtgctgcttcatgtggtcggTTTTTaggcataggacccgtgtct 1702 EGFP0010 LE tcaccagggtgtcgccctTTTTTaggcataggacccgtgtct 1703 EGFP0011 BL cggtggtgcagatgaacttca 1704 EGFP0012 BL catggcggacttgaagaagtc 1705 EGFP0013 BL cgtcctccttgaagtcgatgc 1706

Table 2C shows the alpha-ENaC expression in cynomolgous species for siRNA examples.

Example 3.4 Screening for Interferon-α Induction

To evaluate the ability of siRNA to stimulate interferon-α (IFNα) release, siRNA was incubated with freshly purified peripheral blood mononuclear cells (PBMCs) in vitro for 24 hours. The siRNA was added either directly to PBMCs, or first complexed with a lipid transfection agent (GenePorter 2 or Lipofectamine 2000 or DOTAP transfection agent) and subsequently incubated with PBMCs. As positive controls for IFNα induction, unmodified control sequences DI_A_2216 and DI_A_5167 were included.

DI_A_2216: is a single-stranded antisense DNA  molecule (SEQ ID NO: 1707) 5′-dGsdGsdGdGdGdAdCdGdAdTdCdGdTdCdGsdGsdGsdGs dGsdG -3′ DI_A_5167 is a cholesterol-conjugated siRNA 5′- GUCAUCACACUGAAUACCAAU-s-chol - 3′ (SEQ ID NO: 1708) 3′- CsAsCAGUAGUGUGACUUAUGGUUA-5′

After 24 hours, the IFNα was measured by ELISA. The basal IFNα level was determined for untreated cells and was always very close to a water-only control. The addition of transfection agent alone gave no or little increase of IFNα levels. Known stimulatory oligonucleotides were added to cells, either directly or in the presence of transfectant, and the expected increases of IFNα were observed. This setup allows to determine the stimulation of IFNα in human PBMC by siRNA (or other oligonucleotides).

Isolation of Human PBMCs: A concentrated fraction of leukocytes (buffy coat) was obtained from the Blood Bank Suhl, Institute for Transfusion Medicine, Germany. These cells were negative for a variety of pathogens, including HIV, HCV, and others. The buffy coat was diluted 1:1 with PBS, added to a tube containing Ficoll, and centrifuged for 20 minutes at 2200 rpm to allow fractionation. This was followed by removal of the turbid layer of white blood cells and transferred to a tube with fresh PBS and Ficoll, which was centrifuged for 15 minutes at 2200 rpm. The turbid layer of white blood cells was again removed, transferred to RPMI 1640 culture medium and centrifuged for 5 minutes at 1,200 rpm to pellet the white blood cells. The cells were resuspended in RPMI, pelleted as above, and resuspended in media with 10% FCS to 1×10⁶/mL.

Interferon-α Measurement: Cells in culture were combined with either 500 nM (or 1 μM) oligonucleotide in Optimem or 133 nM oligonucleotide in GP2 or Lipofectamine2000 or DOTAP transfection agent for 24 hours at 37° C. Interferon-α was measured with Bender Med Systems (Vienna, Austria) instant ELISA kit according to the manufacturer's instructions.

Selection of lead therapeutic sequences was based on a level of IFNα induction of less than 15% of the positive control.

Example 3.5 Determination of siRNA Stability in Sputum of CF Patients

Sputum samples were collected by Dr. Ahmet Uluer, Children's Hospital Boston. After collection, sputum samples were treated with antibiotic and were UV-irradiated to reduce bacterial content. To determine siRNA stability in sputum samples, siRNAs were incubated in 30 μL sputum at a concentration of 5 μM at 37° C. for the indicated times. The reaction was terminated by addition of proteinase K and samples were incubated at 42° C. for another 20 minutes. A 40-mer RNA molecule made of L-nucleotides (“Spiegelmer”) resistant to nuclease degradation was added and served as calibration standard. Samples were filtered through a 0.2 μm membrane to remove remaining debris. Samples were analyzed by denaturing ion exchange HPLC on a DNAPac PA 200 column (Dionex) at pH 11.0 using a gradient of sodium perchlorate for elution. siRNAs and degradation products were quantified by determination of the area under the peak for each sample. Concentration was normalized to the concentration in the un-incubated samples.

The selection of the lead therapeutic sequences (ND8356, ND8357 and ND8396) was based on an observed in vitro stability in CF sputum with a half-life greater than 60 minutes.

Example 3.6 Cross-Checking of Lead Therapeutic Sequences Against Known Polymorphisms in Human SCNN1A Gene

To exclude known polymorphisms from the target sites of lead candidates, lead siRNA sequences were checked against the NCBI single nucleotide polymorphism (SNP) database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=snp). Of the 10 known exon polymorphisms in the human SCNN1A gene, none were shown to be present in the target sites of any of the 10 most potent lead therapeutic candidates.

Example 3.7: In Vitro Profiling of Top 5 Predicted Off-Target Sequences

A list of alignments for each sequence was sorted by homology over the 19-mer region. Off-targets were scored based on the number and position of the mismatches in accordance with the criteria described in example 1. The top 5 off-target sequences were identified for each lead therapeutic sequences (ND8356, ND8357 and ND8396). On- and off-target sequences were individually cloned into a dual luciferase reporter system. Each cloned fragment encompassed the target 19 nucleotides in addition to 10 nucleotides flanking region, both 5′ and 3′ of the target sequence. The fragments were cloned into a multiple cloning site 3′ to the Renilla luciferase sequence, under the control of an SV40 promoter. The activity of each siRNA against both on- and off-target sequences was determined by the relative fluorescence of the target Renilla luciferase to the Firefly luciferase, the latter being independently controlled by the HSV-TK promoter. Initially, transfections were performed in COS-7 cells at an siRNA concentration of 50 nM. Luciferase readouts were taken at 24h post-transfection. At this high concentration of siRNA, no knockdown of greater than 30% was observed against any off-target sequence for any of the three lead siRNAs. Activity against the on-target sequence was demonstrated with a relative reduction in Renilla luciferase activity of approximately 80%. IC50 curves were also generated for each siRNA against the on-target sequence and controlled with the off-target sequences identified above. For each lead siRNA, on-target IC50's in this reporter assay were of similar order of magnitude (10-50 μM) to the IC50's obtained against the endogenous target in H441 (Example 3.3) indicating that for ND8356, ND8357 and ND8396, potency against the on-target sequence was at least 1000-5000 fold higher than for any of the predicted off-target sequences.

Example 3.8: Genotoxicity Profiling

Cytotoxicity determination: Cytotoxicity was determined by using a cell counter for the assessment of culture cell number.

It is well known that testing cytotoxic concentrations in vitro may induce genotoxic effects such as micronucleus formation. Therefore, we considered increased numbers of cells containing micronuclei appearing at cell counts of around 50% or less (compared to the concurrent negative control) to be cytotoxicity-related if no dose-dependent increase in micronucleated cells could already be observed at concentrations showing moderate toxicity at most. The analysis of a concentration showing at least 50% reduction in cell count is required by the guidelines regulating in vitro mammalian cell assays (OECD and ICH guidelines for the conduction of chromosome aberration testing). In addition, OECD protocols require testing of non-toxic compounds to include at least one precipitating concentration (as long as this doesn't exceed 10 mM or 5 mg/ml, whichever is lower). Since the in vitro micronucleus test aims to predict the outcome of the regulatory assays, i.e. in vitro chromosomal aberration test, the protocol for the in vitro micronucleus test was designed to meet the requirements for these tests.

Test system: TK6 cells are Ebstein-Barr-Virus transfected and immortalized cells (human lymphoblastoid origin derived from the spleen). Determination of the clastogenic and/or aneugenic potential in the micronucleus test in vitro with TK6 cells with/without S9-liver homogenate (2%) from male rats (Aroclor 1254-pretreated). Treatment time: 20 hr (−S9), 3 hr (+S9). Sampling time: 24 hr after the start of 3-hour treatment, 48 hr after the start of 20-hour treatment. For each substance at least three concentrations (2 cultures per concentration) and 2000 cells per concentration were analyzed.

The micronucleus inducing effect for a tested concentration was considered positive if the frequency of micronucleated cells was

>=2% and showed at least a doubling of the concurrent solvent control value, OR

<2% and showed at least a 3-fold increase over the concurrent solvent control value

To conclude an experiment to be positive, dose-effect relationship and cytotoxicity have to be taken into account.

Summary: Lead therapeutic sequences ND8396, ND8356, ND8357 neither induced increased numbers of cells containing micronuclei after 20-hour treatment without metabolic activation, nor after 3-hour treatment with or without S9. No cytotoxic concentration could be analyzed up to the testing limit of 5 mg/ml.

Example 3.9 In Vitro Functional Efficacy in H441: ND8396

In order to demonstrate in vitro functional efficacy of lead siRNA against alphaENaC H441 cells were transfected with siRNA and prepared for Ussings chamber analysis of ion transport. For transfection, H441 cells were plated into T25 flasks at 2×10⁶ cells per flask in culture medium supplemented with 200 nM Dexamethasone. Cells in each flask were transfected with either ND8396 or a non targeting control siRNA at 30 nM siRNA and 4 mL/mL Lipofectamine 2000 in a total volume of 5 mL (serum free medium). One day after transfection, cells were plated onto 1 cm² Snapwell inserts at confluency (2×10⁵ cells per insert) to minimise the time required for differentiation and formation of tight junctions and supplied with medium in both the apical and the basolateral chambers. After one additional day of culture the apical medium was removed and the basolateral medium replaced, thus taking the cells to air-liquid interface (ALI) culture. Cells were maintained at ALI for a further six days prior to ion transport analysis.

For functional analysis in Ussings chambers, the Snapwell inserts were mounted in Vertical Diffusion Chambers (Costar) and were bathed with continuously gassed Ringer solution (5% CO₂ in O₂; pH 7.4) maintained at 37° C. containing: 120 mM NaCl, 25 mM NaHCO₃, 3.3 mM KH₂PO₄, 0.8 mM K₂HPO₄, 1.2 mM CaCl₂), 1.2 mM MgCl₂, and 10 mM glucose. The solution osmolarity was determined within the range of 280 and 300 mosmol/kgH₂O. Cells were voltage clamped to 0 mV (model EVC4000; WPI). Transepithelial resistance (RT) was measured by applying a 1 or 2-mV pulse at 30-s intervals, or using the initial potential difference across the cells and the initial current measured, and then calculating RT by Ohm's law. Data were recorded using a PowerLab workstation (ADInstruments). Following siRNA treatment the basal characteristics of the cells and the amiloride-sensitive short circuit current (I_(SC) following application of 10 μM amiloride; apical side only) were recorded. ENaC channel activity in each culture was determined by the amiloride-sensitive I_(SC) in each case.

Following assay, cells on the individual inserts were lysed for RNA analysis. A knockdown of 75% at the RNA level at the time of assay (ND8396 as compared to non-targeting control) was correlated with a functional knockdown of the amiloride sensitive current of approximately 30% (ND8396 as compared to non-targeting control).

Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table A.

TABLE A Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A adenosine-5′-phosphate C cytidine-5′-phosphate G guanosine-5′-phosphate T 2′-deoxy-thymidine-5′-phosphate U uridine-5′-phosphate c 2′-O-methylcytidine-5′-phosphate u 2′-O-methyluridine-5′-phosphate Ts 2′-deoxy-thymidine-5′-phosphorothioate

TABLE 1A Selected siRNAs in initial screening set (human-rhesus ENaC alpha cross-reactive siRNAs). A total of 152 iRNA sequences were identified as an initial screening set,  both with (sequence strands 1-304) and without (sequence strands 305-608) backbone modification. iRNA sequences were designed to be fully complementary to both the human and rhesus monkey alpha-ENaC sequences, according to the design criteria described in the examples  section. The percentage residual expression of alpha-ENaC in two independent single-dose transfection experiments is shown (refer to examples section or methods used). 1st screen single dose @ 2nd screen 50 nM in @ 50 nM in Duplex ID Seq ID Sense Seq ID Antissense H441; MV SD H441 SD ND8285 1 AGcccGuAGcGuGGccuccTsT 2 GGAGGCcACGCuACGGGCUTsT 92% 4% 114%  15%  ND8286 3 ccGGGuAAuGGuGcAcGGGTsT 4 CCCGUGcACcAUuACCCGGTsT 60% 1% 84% 4% ND8287 5 AuGcuAucGcGAcAGAAcATsT 6 UGUUCUGUCGCGAuAGcAUTsT 27% 2% 3

% 3% ND8288 7 uGcuAucGcGAcAGAAcAATsT 8 UUGUUCUGUCGCGAuAGcATsT 23% 1% 3

% 4% ND8289 9 GcccGuuuAuGuAuGcuccTsT 10 GGAGcAuAcAuAAACGGGCTsT 64%

% 93% 8% ND8290 11 GcccGuAGcGuGGccuccATsT 12 UGGAGGCcACGCuACGGGCTsT 83% 2% 11

%  6% ND8291 13 ccGGAAAuuAAAGAGGAGcTsT 14 GCUCCUCUUuAAUUUCCGGTsT 54% 2% 79% 1

%  ND8292 15 ccGAAGGuuccGAAGccGATsT 16 UCGGCUUCGGAACCUUCGGTsT 4

% 1% 54% 8% ND8293 17 GcAAuucGGccuGcuuuucTsT 18 GAAAAGcAGGCCGAAUUGCTsT 41% 2% 51% 4% ND8294 19 GGcGAAuuAcucucAcuucTsT 20 GAAGUGAGAGuAAUUCGCCTsT 19% 1% 23%

% ND8295 21 GcGAAuuAcucucAcuuccTsT 22 GGAAGUGAGAGuAAUUCGCTsT 19% 5% 20% 1% ND8296 23 AAccAGGcGAAuuAcucucTsT 24 GAGAGuAAUUCGCCUGCUUTsT 92% 4% 11

%  19%  ND8297 25 GGuAAuGGuGcAcGGGcAGTsT 26 CUGCCCGUGcACcAUuACCTsT 7

% 2% 104%  14%  ND8298 27 cucAcGAuGGcccucGGuGTsT 28 cACCGAGGGCcAUCGUGAGTsT 61% 3% 97% 14%  ND8299 29 GcuccGAAGGuuccGAAGcTsT 30 GCUUCGGAACCUUCGGAGCTsT 16% 2% 19% 3% ND8300 31 GccGAuAcuGGucuccAGGTsT 32 CCUGGAGACcAGuAUCGGCTsT

8% 5% 55% 6% ND8301 33 ccGAuAcuGGucuccAGGcTsT 34 GCCUGGAGACcAGuAUCGGTsT

3% 2% 65% 6% ND8302 35 uGcuGuuGcAccAuAcuuuTsT 36 AAAGuAUGGUGcAAcAGcATsT 19% 1% 25% 3% ND8303 37 AAcGGucuGucccuGAuGcTsT 38 GcAUcAGGGAcAGACCGUUTsT 90% 3% 96% 11%  ND8304 39 uuAAcuuGcGGccuGGcGuTsT 40 ACGCcAGGCCGcAAGUuAATsT 97% 3% 101%  11%  ND8305 41 GcuGGuuAcucAcGAuGGcTsT 42 GCcAUCGUGAGuAACcAGCTsT 73% 2% 78% 7% ND8306 43 uuAcucAcGAuGGcccucGTsT 44 CGAGGGCcAUCGUGAGuAATsT 91% 7% 83% 6% ND8307 45 GAAGccGAuACuGGucuccTsT 46 GGAGACcAGuAUCGGCUUCTsT 71% 3% 73% 6% ND8308 47 GAuAcuGGucuccAGGccGTsT 48 CGGCCUGGAGACcAGuAUCTsT 86% 1% 90% 9% ND8309 49 AuAcuGGucuccAGGccGATsT 50 UCGGCCUGGAGACcAGuAUTsT 71% 5% 70% 8% ND8310 51 cAAcGGucuGucccuGAuGTsT 52 cAUcAGGGAcAGACCGUUGTsT 80% 2% 84% 9% ND8311 53 uuuAAcuuGcGGccuGGcGTsT 54 CGCcAGGCCGcAAGUuAAATsT 96% 2% 107%  1

%  ND8312 55 uAcucAcGAuGGcccucGGTsT 56 CCGAGGGCcAUCGUGAGuATsT 44% 2% 97% 9% ND8313 57 uuucGGAGAGuAcuucAGcTsT 58 GCUGAAGuACUCUCCGAAATsT 14% 2% 16% 2% ND8314 59 GcAGAcGcucuuuGAccuGTsT 60 cAGGUcAAAGAGCGUCUGCTsT 55% 4% 58% 5% ND8315 61 cuAcAucuucuAuccGcGGTsT 62 CCGCGGAuAGAAGAUGuAGTsT 20% 3% 26% 4% ND8316 63 AGGcGAAuuAcucucAcuuTsT 64 AAGUGAGAGuAAUUCGCCUTsT 24% 1% 25% 2% ND8317 65 ccGcuucAAccAGGucuccTsT 66 GGAGACCUGGUUGAAGCGGTsT 62% 6% 64% 4% ND8318 67 cAAccGcAuGAAGAcGGccTsT 68 GGCCGUCUUcAUGCGGUUGTsT 54% 6% 54% 4% ND8319 69 AuGAAGAcGGccuucuGGGTsT 70 CCcAGAAGGCCGUCUUcAUTsT 44% 4% 44% 6% ND8320 71 AGcAcAAccGcAuGAAGAcTsT 72 GUCUUcAUGCGGUUGUGCUTsT 15% 1% 16% 1% ND8321 73 ucGAGuuccAccGcuccuATsT 74 uAGGAGCGGUGGAACUCGATsT 86% 6% 89% 13%  ND8322 75 cuGcuucuAccAGAcAuAcTsT 76 GuAUGUCUGGuAGAAGcAGTsT 46% 4% 44% 3% ND8323 77 GAGGGAGuGGuAccGcuucTsT 78 GAAGCGGuACcACUCCCUCTsT 60% 7% 56% 3% ND8324 79 ccuuuAuGGAuGAuGGuGGTsT 80 CcACcAUcAUCcAuAAAGGTsT 83% 9% 83% 1% ND8325 81 uGAGGGAGuGGuAccGcuuTsT 82 AAGCGGuACcACUCCCUcATsT 77% 6% 72% 2% ND8326 83 ccuGcAAccAGGcGAAuuATsT 84 uAAUUCGCCUGGUUGcAGGTsT 41% 4% 44% 7% ND8327 85 GGccuGGcGuGGAGAccucTsT 86 GAGGUCUCcACGCcAGGCCTsT 101%  5% 96% 4% ND8328 87 uGcuuuucGGAGAGuAcuuTsT 88 AAGuACUCUCCGAAAAGcATsT 36% 1% 39% 3% ND8329 89 cccGuAGcGuGGccuccAGTsT 90 CUGGAGGCcACGCuACGGGTsT 52% 1% 51% 2% ND8330 91 ccGuAGcGuGGccuccAGcTsT 92 GCUGGAGGCcACGCuACGGTsT 86% 9% 84% 3% ND8331 93 ccAGGcGAAuuAcucucAcTsT 94 GUGAGAGuAAUUCGCCUGGTsT 16% 2% 13% 1% ND8332 95 GAAAcuGcuAuAcuuucAATsT 96 UUGAAAGuAuAGcAGUUUCTsT 10% 1% 10% 1% ND8333 97 GcccGGGuAAuGGuGcAcGTsT 98 CGUGcACcAUuACCCGGGCTsT 93% 6% 82% 4% ND8334 99 cccGGGuAAuGGuGcAcGGTsT 100 CCGUGcACcAUuACCCGGGTsT 96% 4% 71% 10%  ND8335 101 cGGGuAAuGGuGcAcGGGcTsT 102 GCCCGUGcACcAUuACCCGTsT 42% 3% 91% 8% ND8336 103 GGGuAAuGGuGcAcGGGcATsT 104 UGCCCGUGcACcAUuACCCTsT 65% 5% 71% 7% ND8337 105 uAAuGGuGcAcGGGcAGGATsT 106 UCCUGCCCGUGcACcAUuATsT 46% 3% 46% 4% ND8338 107 cuGGuuAcucAcGAuGGccTsT 108 GGCcAUCGUGAGuAACcAGTsT 74% 5% 79% 10%  ND8339 109 GuuAcucAcGAuGGcccucTsT 110 GAGGGCcAUCGUGAGuAACTsT 85% 6% 92% 8% ND8340 111 uGucAcGAuGGucAcccucTsT 112 GAGGGUGACcAUCGUGAcATsT 85% 4% 74% 5% ND8341 113 uGcuccGAAGGuuccGAAGTsT 114 CUUCGGAACCUUCGGAGcATsT 37% 2% 32% 3% ND8342 115 uccGAAGGuuccGAAGccGTsT 116 CGGCUUCGGAACCUUCGGATsT 60% 4% 47% 5% ND8343 117 uuccGAAGccGAuAcuGGuTsT 118 ACcAGuAUCGGCUUCGGAATsT 1

% 1% 1

% 2% ND8344 119 AGccGAuAcuGGucuccAGTsT 120 CUGGAGACcAGUAUCGGCUTsT 49% 3% 41% 3% ND8345 121 cuuGGuAcuGccucuGAAcTsT 122 GUUcAGAGGcAGuACcAAGTsT 55% 2% 47% 4% ND8346 123 cucccGuAGcAcAcuAuAATsT 124 UuAuAGUGUGCuACGGGAGTsT 67% 3% 67%

% ND8347 125 ucccGuAGcAcAcuAuAAcTsT 126 GUuAuAGUGUGCuACGGGATsT 29% 1%

6% 3% ND8348 127 uGcAccAuAcuuucuuGuATsT 128 uAcAAGAAAGuAUGGUGcATsT 17% 1% 15% 3% ND8349 129 uuGcccGuuuAuGuAuGcuTsT 130 AGcAuAcAuAAACGGGcAATsT 68% 2% 60% 4% ND8350 131 uGcccGuuuAuGuAuGcucTsT 132 GAGcAuAcAuAAACGGCcATsT

9% 8% 44% 6% ND8351 133 GGAcccuAGAccucuGcAGTsT 134 CUGcAGAGGUCuAGGGUCCTsT 86% 11%  82% 2% ND8352 135 ccuAGAccucuGcAGcccATsT 136 UGGGCUGcAGAGGUCuAGGTsT 69% 7% 79% 3% ND8353 137 uGGcAuGAuGuAcuGGcAATsT 138 UUGCcAGuAcAUcAUGCcATsT 58% 4% 52% 4% ND8354 139 uAcuGGcAAuucGGccuGcTsT 140 GcAGGCCGAAUUGCcAGuATsT 101%  4% 100%  4% ND8355 141 AAuucGGccuGcuuuucGGTsT 142 CCGAAAAGcAGGCCGAAUUTsT 49% 1% 4

% 6% ND8356 143 cuGcuuuucGGAGAGuAcuTsT 144 AGuACUCUCCGAAAAGcAGTsT 17% 3% 18% 1% ND8357 145 uucGGAGAGuAcuucAGcuTsT 146 AGCUGAAGuACUCUCCGAATsT 13% 3% 16% 2% ND8358 147 AGcAGAcGcucuuuGAccuTsT 148 AGGUcAAAGAGCGUCUGCUTsT 73% 9% 71% 5% ND8359 149 cuuGcAGcGccuGAGGGucTsT 150 GACCCUcAGGCGCUGcAAGTsT 57% 9% 64% 7% ND8360 151 uGGcuuuAAcuuGcGGccuTsT 152 AGGCCGcAAGUuAAAGCcATsT 102%  12%  106%  10%  ND8361 153 GcuuuAAcuuGcGGccuGGTsT 154 CcAGGCCGcAAGUuAAAGCTsT 83%

% 82% 8% ND8362 155 uAAcuuGcGGccuGGcGuGTsT 156 cACGCcAGGCCGcAAGUuATsT 119%  3% 115%  6% ND8363 157 AccuuuAcccuucAAAGuATsT 158 uACUUUGAAGGGuAAAGGUTsT 17% 3% 13% 2% ND8364 159 GGuuAcucAcGAuGGcccuTsT 160 AGGGCcAUCGUGAGuAACCTsT 104%  9% 117%  17%  ND8365 161 cAcGAuGGcccucGGuGAcTsT 162 GUcACCGAGGGCcAUCGUGTsT 140%  13%  100%  9% ND8366 163 AGAuGcuAucGcGAcAGAATsT 164 UUCUGUCGCGAuAGcAUCUTsT 46% 2% 70% 6% ND8367 165 AcGAuGGucAcccuccuGuTsT 166 AcAGGAGGGUGACcAUCGUTsT 85% 6% 128%  10%  ND8368 167 cuccGAAGGuuccGAAGccTsT 168 GGCUUCGGAACCUUCGGAGTsT 12% 2% 18% 1% ND8369 169 AAGGuuccGAAGccGAuAcTsT 170 GuAUCGGCUUCGGAACCUUTsT 63% 7% 114%  19%  ND8370 171 GGuAcuGccucuGAAcAcuTsT 172 AGUGUUcAGAGGcAGuACCTsT 36% 3% 71% 6% ND8371 173 AGcuuuGAcAAGGAAcuuuTsT 174 AAAGUUCCUUGUcAAAGCUTsT 17% 1% 21% 1% ND8372 175 uuuGAcAAGGAAcuuuccuTsT 176 AGGAAAGUUCCUUGUcAAATsT 16% 2% 26% 4% ND8373 177 uGAcAAGGAAcuuuccuAATsT 178 UuAGGAAAGUUCCUUGUcATsT 12% 1% 22% 5% ND8374 179 cccGuAGcAcAcuAuAAcATsT 180 UGUuAuAGUGUGCuACGGGTsT 41% 2% 75% 3% ND8375 181 cAcuAuAAcAucuGcuGGATsT 182 UCcAGcAGAUGUuAuAGUGTsT 17% 1% 26% 2% ND8376 183 uuGcuGuuGcAccAuAcuuTsT 184 AAGuAUGGUGcAAcAGcAATsT 45% 4% 69% 6% ND8377 185 GuAcuGGcAAuucGGccuGTsT 186 cAGGCCGAAUUGCcAGuACTsT 60% 6% 120%  8% ND8378 187 uucGGccuGcuuuucGGAGTsT 188 CUCCGAAAAGcAGGCCGAATsT 57%

% 86% 11%  ND8379 189 ccuGcuuuucGGAGAGuACTsT 190 GuACUCUCCGAAAAGcAGGTsT 43%

% 50% 3% ND8380 191 GcuuuucGGAGAGuAcuucTsT 192 GAAGuACUCUCCGAAAAGCTsT 16% 2% 24% 2% ND8381 193 cuuuucGGAGAGuAcuucATsT 194 UGAAGuACUCUCCGAAAAGTsT 12% 1% 16% 3% ND8382 195 cAAccucAAcucGGAcAAGTsT 196 CUUGUCCGAGUUGAGGUUGTsT 33% 2% 39% 3% ND8383 197 cuAccAGAcAuAcucAucATsT 198 UGAUGAGuAUGUCUGGuAGTsT 13% 1% 23% 6% ND8384 199 cuGucGAGGcuGccAGAGATsT 200 UCUCUGGcAGCCUCGAcAGTsT 11% 1% 18% 3% ND8385 201 AAAcuGcuAuAcuuucAAuTsT 202 AUUGAAAGuAuAGcAGUUUTsT 48% 8% 64% 11%  ND8386 203 GGcuuuAAcuuGcGGccuGTsT 204 cAGGCCGcAAGUuAAAGCCTsT 55% 7% 70% 8% ND8387 205 cuuuAAcuuGcGGccuGGcTsT 206 GCcAGGCCGcAAGUuAAAGTsT 40% 11%  87% 14%  ND8388 207 AGGuGuGuAuucAcuccuGTsT 208 cAGGAGUGAAuAcAcACCUTsT 45% 3% 41% 5% ND8389 209 AcGAuGGcccucGGuGAcATsT 210 UGUcACCGAGGGCcAUCGUTsT 43% 2% 60% 9% ND8390 211 cuGAAcAcucuGGuuucccTsT 212 GGGAAACcAGAGUGUUcAGTsT 33% 1% 48% 11%  ND8391 213 cuAuAAcAucuGcuGGAGuTsT 214 ACUCcAGcAGAUGUuAuAGTsT 16% 1% 17% 4% ND8392 215 GcAccAuAcuuucuuGuAcTsT 216 GuAcAAGAAAGuAUGGUGCTsT 19% 1% 22% 4% ND8393 217 uGucuAGcccAucAuccuGTsT 218 cAGGAUGAUGGGCuAGAcATsT 6

% 3% 92% 15%  ND8394 219 AGGAcccuAGAccucuGcATsT 220 UGcAGAGGUCuAGGGUCCUTsT 94% 6% 86% 13%  ND8395 221 ccAccGcuccuAccGAGAGTsT 222 CUCUCGGuAGGAGCGGUGGTsT 55% 1% 65% 6% ND8396 223 uAccGAGAGcucuucGAGuTsT 224 ACUCGAAGAGCUCUCGGuATsT 11% 1% 11% 1% ND8397 225 AAcAuccuGucGAGGcuGcTsT 226 GcAGCCUCGAcAGGAUGUUTsT 90% 7% 72% 11%  ND8398 227 GAAccuuuAcccuucAAAGTsT 228 CUUUGAAGGGuAAAGGUUCTsT 22% 2% 25% 4% ND8399 229 GGuuccGAAGccGAuAcuGTsT 230 cAGuAUCGGCUUCGGAACCTsT 93% 9% 89% 9% ND8400 231 AAGccGAuAcuGGucuccATsT 232 UGGAGACcAGuAUCGGCUUTsT 3

% 2% 42% 9% ND8401 233 ucuAGcccAucAuccuGcuTsT 234 AGcAGGAUGAUGGGCuAGATsT 9

% 8% 95% 14%  ND8402 235 cGGcGccAuccGccuGGuGTsT 236 cACcAGGCGGAUGGCGCCGTsT 81% 8% 89% 17%  ND8403 237 uuuucGGAGAGuAcuucAGTsT 238 CUGAAGuACUCUCCGAAAATsT 13% 1% 13% 1% ND8404 239 GAGAGuAcuucAGcuAcccTsT 240 GGGuAGCUGAAGuACUCUCTsT 71% 3% 100%  10%  ND8405 241 GAcGcucuuuGAccuGuAcTsT 242 GuAcAGGUcAAAGAGCGUCTsT 84% 5% 92% 13%  ND8406 243 uGuGuAuucAcuccuGcuuTsT 244 AAGcAGGAGUGAAuAcAcATsT 78% 2% 89% 8% ND8407 245 AAcAAcAAGAGAAAuGGAGTsT 246 CUCcAUUUCUCUUGUUGUUTsT 66% 3% 88% 21%  ND8408 247 AuuGAAGGAuGuGcAGGGcTsT 248 GCCCUGcAcAUCCUUcAAUTsT 25% 1% 36% 6% ND8409 249 ucucAGAGccGcccAAAcuTsT 250 AGUUUGGGCGGCUCUGAGATsT 18% 1% 24% 2% ND8410 251 AAAcAcAAccAAGGGuAcATsT 252 UGuACCCUUGGUUGUGUUUTsT 21% 1% 35% 2% ND8411 253 uAcccGuGcccucAcAGAGTsT 254 CUCUGUGAGGGcACGGGuATsT 57% 2% 67% 4% ND8412 255 uAGcAcAcuAuAAcAucuGTsT 256 cAGAUGUuAuAGUGUGCuATsT 30% 2% 41% 1% ND8413 257 GGuGuGuAuucAcuccuGcTsT 258 GcAGGAGUGAAuAcAcACCTsT 73% 1% 90% 9% ND8414 259 cAuGAucAAGGAGuGuGGcTsT 260 GCcAcACUCCUUGAUcAUGTsT 65% 2% 67% 5% ND8415 261 AcucAcGAuGGcccucGGuTsT 262 ACCGAGGGCcAUCGUGAGUTsT 96% 6% 95% 6% ND8416 263 GGAGcuuuGAcAAGGAAcuTsT 264 AGUUCCUUGUcAAAGCUCCTsT 24% 1% 28% 4% ND8417 265 AuAcccGuGcccucAcAGATsT 266 UCUGUGAGGGcACGGGuAUTsT 54% 1% 62% 2% ND8418 267 GGAGuGGccAAAGucAAcATsT 268 UGUUGACUUUGGCcACUCCTsT 93% 2% 86% 11%  ND8419 269 AAcuAcAAAAccAAuucuGTsT 270 cAGAAUUGGUUUUGuAGUUTsT 101%  5% 108%  19%  ND8420 271 uGcuGGAGuGuuGcuGuuGTsT 272 cAAcAGcAAcACUCcAGcATsT 29% 1% 26% 1% ND8421 273 AGGucuccuGcAAccAGGcTsT 274 GCCUGGUUGcAGGAGACCUTsT 95% 10%  91% 17%  ND8422 275 cuuuGGcAuGAuGuAcuGGTsT 276 CcAGuAcAUcAUGCcAAAGTsT 86% 3% 84% 6% ND8423 277 cAucuGcAcccucAAucccTsT 278 GGGAUUGAGGGUGcAGAUGTsT 82% 11%  73% 4% ND8424 279 cGAcuGcAccAAGAAuGGcTsT 280 GCcAUUCUUGGUGcAGUCGTsT 70% 8% 69% 7% ND8425 281 AAAAcAcAAccAAGGGuAcTsT 282 GuACCCUUGGUUGUGUUUUTsT 95% 6% 106%  13%  ND8426 283 cAucuGcuGGAGuGuuGcuTsT 284 AGcAAcACUCcAGcAGAUGTsT 30% 3% 37% 1% ND8427 285 ccuAcAucuucuAuccGcGTsT 286 CGCGGAuAGAAGAUGuAGGTsT 42% 6% 30% 1% ND8428 287 GccuAcAucuucuAuccGcTsT 288 GCGGAuAGAAGAUGuAGGCTsT 65% 7%

4% 3% ND8429 289 GAGuGGuAccGcuuccAcuTsT 290 AGUGGAAGCGGuACcACUCTsT 95% 11%  86% 19%  ND8430 291 GGuAccGcuuccAcuAcAuTsT 292 AUGuAGUGGAAGCGGuACCTsT 111%  19%  96% 14%  ND8431 293 GuGGuAccGcuuccAcuAcTsT 294 GuAGUGGAAGCGGuACcACTsT 98% 13% 

2% 26%  ND8432 295 GAAuuAcucucAcuuccAcTsT 296 GUGGAAGUGAGAGuAAUUCTsT 111%  21%  73% 27%  ND8433 297 AAuuAcucucAcuuccAccTsT 298 GGUGGAAGUGAGAGuAAUUTsT 109%  22%  105%  7% ND8434 299 uAcucucAcuuccAccAccTsT 300 GGUGGUGGAAGUGAGAGuATsT 106%  23%  95% 7% ND8435 301 AGuGGuAccGcuuccAcuATsT 302 uAGUGGAAGCGGuACcACUTsT 109%  18%  102%  9% ND8436 303 GGGcAAcuucAucuucGccTsT 304 GGCGAAGAUGAAGUUGCCCTsT 109%  18%  107%  14%  ND-8501 305 AGCCCGUAGCGUGGCCUCCTsT 306 GGAGGCCACGCUACGGGCUTsT 84% 14%  69% 3% ND-8502 307 CCGGGUAAUGGUGCACGGGTsT 308 CCCGUGCACCAUAACCCGGTsT 41% 6% 30% 2% ND-8503 309 AUGCUAUCGCGACAGAACATsT 310 UGUUCUGUCGCGAUAGCAUTsT 11% 2% 10% 0% ND-8504 311 UGCUAUCGCGACAGAACAATsT 312 UUGUUCUGUCGCGAUAGCATsT 1

% 2% 10% 1% ND-8505 313 GCCCGUUUAUGUAUGCUCCTsT 314 GGAGCAUACAUAAACGGGCTsT 23% 3% 16% 1% ND-8506 315 GCCCGUAGCGUGGCCUCCATsT 316 UGGAGGCCACGCUACGGGCTsT 32% 3% 22% 1% ND-8507 317 CCGGAAAUUAAAGAGGAGCTsT 318 GCUCCUCUUUAAUUUCCGGTsT 35% 4% 24% 1% ND-8508 319 CCGAAGGUUCCGAAGCCGATsT 320 UCGGCUUCGGAACCUUCGGTsT 19% 2% 13% 1% ND-8509 321 GCAAUUCGGCCUGCUUUUCTsT 322 GAAAAGCAGGCCGAAUUGCTsT 12% 1%  8% 1% ND-8510 323 GGCGAAUUACUCUCACUUCTsT 324 GAAGUGAGAGUAAUUCGCCTsT 21% 2% 18% 1% ND-8511 325 GCGAAUUACUCUCACUUCCTsT 326 GGAAGUGAGAGUAAUUCGCTsT 12% 2%  8% 1% ND-8512 327 AACCAGGCGAAUUACUCUCTsT 328 GAGAGUAAUUCGCCUGGUUTsT 99% 11%  79% 5% ND-8513 329 GGUAAUGGUGCACGGGCAGTsT 330 CUGCCCGUGCACCAUUACCTsT 61% 6% 42% 4% ND-8514 331 CUCACGAUGGCCCUCGGUGTsT 332 CACCGAGGGCCAUCGUGAGTsT 94% 11%  70% 4% ND-8515 333 CGUCCGAAGGUUCCGAAGCTsT 334 GCUUCGGAACCUUCGGAGCTsT 18% 2% 17% 2% ND-8516 335 GCCGAUACUGGUCUCCAGGTsT 336 CCUGGAGACCAGUAUCGGCTsT 14% 1% 12% 1% ND-8517 337 CCGAUACUGGUCUCCAGGCTsT 338 GCCUGGAGACCAGUAUCGGTsT 42% 5% 33% 2% ND-8518 339 UGCUGUUGCACCAUACUUUTsT 340 AAAGUAUGGUGCAACAGCATsT 10% 1%  9% 0% ND-8519 341 AACGGUCUGUCCCUGAUGCTsT 342 GCAUCAGGGACAGACCGUUTsT 60% 7% 52% 8% ND-8520 343 UUAACUUGCGGCCUGGCGUTsT 344 ACGCCAGGCCGCAAGUUAATsT 82% 25%  77% 18%  ND-8521 345 GCUGGUUACUCACGAUGGCTsT 346 GCCAUCGUGAGUAACCAGCTsT 36% 4% 34% 7% ND-8522 347 UUACUCACGAUGGCCCUCGTsT 348 CGAGGGCCAUCGUGAGUAATsT 105%  21%  113%  21%  ND-8523 349 GAAGCCGAUACUGGUCUCCTsT 350 GGAGACCAGUAUCGGCUUCTsT 24% 2% 18% 2% ND-8524 351 GAUACUGGUCUCCAGGCCGTsT 352 CGGCCUGGAGACCAGUAUCTsT 30% 5% 25% 3% ND-8525 353 AUACUGGUCUCCAGGCCGATsT 354 UCGGCCUGGAGACCAGUAUTsT 12% 1% 11% 2% ND-8526 355 CAACGGUCUGUCCCUGAUGTsT 356 CAUCAGGGACAGACCGUUGTsT

4% 7% 24% 2% ND-8527 357 UUUAACUUGCGGCCUGGCGTsT 358 CGCCAGGCCGCAAGUUAAATsT 122%  6% 107%  9% ND-8528 359 UACUCACGAUGGCCCUCGGTsT 360 CCGAGGGCCAUCGUGAGUATsT 78% 6% 84% 7% ND-8529 361 UUUCGGAGAGUACUUCAGCTsT 362 GCUGAAGUACUCUCCGAAATsT 87% 18%  80% 17%  ND-8530 363 GCAGACGCUCUUUGACCUGTsT 364 CAGGUCAAAGAGCGUCUGCTsT 14% 2% 13% 0% ND-8531 365 CUACAUCUUCUAUCCGCGGTsT 366 CCGCGGAUAGAAGAUGUAGTsT 20% 4% 18% 3% ND-8532 367 AGGCGAAUUACUCUCACUUTsT 368 AAGUGAGAGUAAUUCGCCUTsT 2

% 5% 18% 1% ND-8533 369 CCGCUUCAACCAGGUCUCCTsT 370 GGAGACCUGGUUGAAGCGGTsT 30% 11%  22% 2% ND-8534 371 CAACCGCAUGAAGACGGCCTsT 372 GGCCGUCUUCAUGCGGUUGTsT 33% 4% 23% 1% ND-8535 373 AUGAAGACGGCCUUCUGGGTsT 374 CCCAGAAGGCCGUCUUCAUTsT 114%  12%  84% 15%  ND-8536 375 AGCACAACCGCAUGAAGACTsT 376 GUCUUCAUGCGGUUGUGCUTsT 18% 1% 16% 3% ND-8537 377 UCGAGUUCCACCGCUCCUATsT 378 UAGGAGCGGUGGAACUCGATsT 25% 0% 26% 3% ND-8538 379 CUGCUUCUACCAGACAUACTsT 380 GUAUGUCUGGUAGAAGCAGTsT 12% 1% 13% 2% ND-8539 381 GAGGGAGUGGUACCGCUUCTsT 382 GAAGCGGUACCACUCCCUCTsT 43% 1% 47% 25%  ND-8540 383 CCUUUAUGGAUGAUGGUGGTsT 384 CCACCAUCAUCCAUAAAGGTsT 61% 5% 60% 8% ND-8541 385 UGAGGGAGUGGUACCGCUUTsT 386 AAGCGGUACCACUCCCUCATsT 36% 5% 35% 5% ND-8542 387 CCUGCAACCAGGCGAAUUATsT 388 UAAUUCGCCUGGUUGCAGGTsT 19% 2% 16% 1% ND-8543 389 GGCCUGGCGUGGAGACCUCTsT 390 GAGGUCUCCACGCCAGGCCTsT 28% 7% 20% 2% ND-8544 391 UGCUUUUCGGAGAGUACUUTsT 392 AAGUACUCUCCGAAAAGCATsT 22%

% 17% 1% ND-8545 393 CCCGUAGCGUGGCCUCCAGTsT 394 CUGGAGGCCACGCUACGGGTsT 25% 3% 22% 3% ND-8546 395 CCGUAGCGUGGCCUCCAGCTsT 396 GCUGGAGGCCACGCUACGGTsT 62% 5% 57% 9% ND-8547 397 CCAGGCGAAUUACUCUCACTsT 398 GUGAGAGUAAUUCGCCUGGTsT 83% 11%  16% 2% ND-8548 399 GAAACUGCUAUACUUUCAATsT 400 UUGAAAGUAUAGCAGUUUCTsT  9% 3%  3% 0% ND-8549 401 GCCCGGGUAAUGGUGCACGTsT 402 CGUGCACCAUUACCCGGGCTsT 87% 9% 92% 14%  ND-8550 403 CCCGGGUAAUGGUGCACGGTsT 404 CCGUGCACCAUUACCCGGGTsT 19% 12%  14% 1% ND-8551 405 CGGGUAAUGGUGCACGGGCTsT 406 GCCCGUGCACCAUUACCCGTsT 68% 11%  73% 3% ND-8552 407 GGGUAAUGGUGCACGGGCATsT 408 UGCCCGUGCACCAUUACCCTsT 30% 6% 33% 2% ND-8553 409 UAAUGGUGCACGGGCAGGATsT 410 UCCUGCCCGUGCACCAUUATsT 29% 3% 31% 1% ND-8554 411 CUGGUUACUCACGAUGGCCTsT 412 GGCCAUCGUGAGUAACCAGTsT 74% 16%  66% 8% ND-8555 413 GUUACUCACGAUGGCCCUCTsT 414 GAGGGCCAUCGUGAGUAACTsT 91% 21%  88% 10%  ND-8556 415 UGUCACGAUGGUCACCCUCTsT 416 GAGGGUGACCAUCGUGACATsT 72% 4% 76% 12%  ND-8557 417 UGCUCCGAAGGUUCCGAAGTsT 418 CUUCGGAACCUUCGGAGCATsT 51% 2%

9% 18%  ND-8558 419 UCCGAAGGUUCCGAAGCCGTsT 420 CGGCUUCGGAACCUUCGGATsT 109%  11%  77% 13%  ND-8559 421 UUCCGAAGCCGAUACUGGUTsT 422 ACCAGUAUCGGCUUCGGAATsT 46% 20%  33% 6% ND-8560 423 AGCCGAUACUGGUCUCCAGTsT 424 CUGGAGACCAGUAUCGGCUTsT 15% 6% 10% 1% ND-8561 425 CUUGGUACUGCCUCUGAACTsT 426 CUUCAGAGGCAGUACCAAGTsT 16% 3% 12% 3% ND-8562 427 CUCCCGUAGCACACUAUAATsT 428 UUAUAGUGUGCUACGGGAGTsT 14% 6% 10% 1% ND-8563 429 UCCCGUAGCACACUAUAACTsT 430 GUUAUAGUGUGCUACGGGATsT 43% 11%  36% 4% ND-8564 431 UGCACCAUACUUUCUUGUATsT 432 UACAAGAAAGUAUGGUGCATsT 17% 6% 13% 3% ND-8565 433 UUGCCCGUUUAUGUAUGCUTsT 434 AGCAUACAUAAACGGGCAATsT 84% 2% 103%  12%  ND-8566 435 UGCCCGUUUUAUGUAGCUCTsT 436 GAGCAUACAUAAACGGGCATsT 69% 25%  93% 4% ND-8567 437 GGACCCUAGACCUCUGCAGTsT 438 CUGCAGAGGUCUAGGGUCCTsT 29% 8% 33% 2% ND-8568 439 CCUAGACCUCUGCAGCCCATsT 440 UGGGCUGCAGAGGUCUAGGTsT 18% 2% 19% 1% ND-8569 441 UGGCAUGAUGUACUGGCAATsT 442 UUGCCAGUACAUCAUGCCATsT 19% 3% 20% 5% ND-8570 443 UACUGGCAAUUCGGCCUGCTsT 444 GCAGGCCGAAUUGCCAGUATsT 86% 15%  83% 16%  ND-8571 445 AAUUCGGCCUGCUUUUCGGTsT 446 CCGAAAAGCAGGCCGAAUUTsT 19% 3% 24% 4% ND-8572 447 CUGCUUUUCGGAGAGUACUTsT 448 AGUACUCUCCGAAAAGCAGTsT  8% 2% 12% 2% ND-8573 449 UUCGGAGAGUACUUCAGCUTsT 450 AGCUGAAGUACUCUCCGAATsT 27% 3% 40% 5% ND-8574 451 AGCAGACGCUCUUUGACCUTsT 452 AGGUCAAAGAGCGUCUGCUTsT 15% 0% 19% 4% ND-8575 453 CUUGCAGCGCCUGAGGGUCTsT 454 GACCCUCAGGCGCUGCAAGTsT 35% 1% 40% 4% ND-8576 455 UGGCUUUAACUUGCGGCCUTsT 456 AGGCCGCAAGUUAAAGCCATsT 47% 3% 53% 8% ND-8577 457 GCUUUAACUUGCGGCCUGGTsT 458 CCAGGCCGCAAGUUAAAGCTsT 20% 2% 25% 5% ND-8578 459 UAACUUGCGGCCUGGCGUGTsT 460 CACGCCAGGCCGCAAGUUATsT 75% 7% 82% 4% ND-8579 461 ACCUUUACCCUUCAAAGUATsT 462 UACUUUGAAGGGUAAAGGUTsT 14% 2% 17% 3% ND-8580 463 GGUUACUCACGAUGGCCCUTsT 464 AGGGCCAUCGUGAGUAACCTsT 63% 5% 70% 11%  ND-8581 465 CACGAUGGCCCUCGGUGACTsT 466 GUCACCGAGGGCCAUCGUGTsT 56% 2% 60% 6% ND-8582 467 AGAUGCUAUCGCGACAGAATsT 468 UUCUGUCGCGAUAGCAUCUTsT 18% 1% 18% 1% ND-8583 469 ACGAUGGUCACCCUCCUGUTsT 470 ACAGGAGGGUGACCAUCGUTsT 48% 3% 62% 6% ND-8584 471 CUCCGAAGGUUCCGAAGCCTsT 472 GGCUUCGGAACCUUCGGAGTsT 18% 2% 20% 5% ND-8585 473 AAGGUUCCGAAGCCGAUACTsT 474 GUAUCGGCUUCGGAACCUUTsT 26% 3% 28% 1% ND-8586 475 GGUACUGCCUCUGAACACUTsT 476 AGUGUUCAGAGGCAGUACCTsT 12% 2% 12% 1% ND-8587 477 AGCUUUGACAAGGAACUUUTsT 478 AAAGUUCCUUGUCAAAGCUTsT 17% 1% 18% 2% ND-8588 479 UUUGACAAGGAACUUUCCUTsT 480 AGGAAAGUUCCUUGUCAAATsT 78% 2% 73% 2% ND-8589 481 UGACAAGGAACUUUCCUAATsT 482 UUAGGAAAGUUCCUUGUCATsT 14% 5% 16% 1% ND-8590 483 CCCGUAGCACACUAUAACATsT 484 UGUUAUAGUGUGCUACGGGTsT  9% 1% 11% 2% ND-8591 485 CACUAUAACAUCUGCUGGATsT 486 UCCAGCAGAUGUUAUAGUGTsT 18% 2% 20% 2% ND-8592 487 UUGCUGUUGCACCAUACUUTsT 488 AAGUAUGGUGCAACAGCAATsT 23% 2% 26% 8% ND-8593 489 GUACUGGCAAUUCGGCCUGTsT 490 CAGGCCGAAUUGCCAGUACTsT 66% 3% 62% 4% ND-8594 491 UUCGGCCUGCUUUUCGGAGTsT 492 CUCCGAAAAGCAGGCCGAATsT 97% 7% 86% 8% ND-8595 493 CCUGCUUUUCGGAGAGUACTsT 494 GUACUCUCCGAAAAGCAGGTsT 11% 3% 14% 3% ND-8596 495 GCUUUUCGGAGAGUACUUCTsT 496 GAAGUACUCUCCGAAAAGCTsT 12% 1% 17% 2% ND-8597 497 CUUUUCGGAGAGUACUUCATsT 498 UGAAGUACUCUCCGAAAAGTsT 11% 1% 14% 2% ND-8598 499 CAACCUCAACUCGGACAAGTsT 500 CUUGUCCGAGUUGAGGUUGTsT 15% 2% 16% 2% ND-8599 501 CUACCAGACAUACUCAUCATsT 502 UGAUGAGUAUGUCUGGUAGTsT 17% 1% 18% 2% ND-8600 503 CUGUCGAGGCUGCCAGAGATsT 504 UCUCUGGCAGCCUCGACAGTsT 17% 0% 16% 1% ND-8601 505 AAACUGCUAUACUUUCAAUTsT 506 AUUGAAAGUAUAGCAGUUUTsT 28% 1% 26% 1% ND-8602 507 GGCUUUAACUUGCGGCCUGTsT 508 CAGGCCGCAAGUUAAAGCCTsT 21% 2% 18% 1% ND-8603 509 CUUUAACUUGCGGCCUGGCTsT 510 GCCAGGCCGCAAGUUAAAGTsT 81% 2% 69% 6% ND-8604 511 AGGUGUGUAUUCACUCCUGTsT 512 CAGGAGUGAAUACACACCUTsT 47% 4% 40% 1% ND-8605 513 ACGAUGGCCCUCGGUGACATsT 514 UGUCACCGAGGGCCAUCGUTsT 40% 6% 35% 2% ND-8606 515 CUGAACACUCUGGUUUCCCTsT 516 GGGAAACCAGAGUGUUCAGTsT 60% 2% 75% 4% ND-8607 517 CUAUAACAUCUGCUGGAGUTsT 518 ACUCCAGCAGAUGUUAUAGTsT 17% 1% 24% 3% ND-8608 519 GCACCAUACUUUCUUGUACTsT 520 GUACAAGAAAGUAUGGUGCTsT 10% 1% 15% 3% ND-8609 521 UGUCUAGCCCAUCAUCCUGTsT 522 CAGGAUGAUGGGCUAGACATsT 62% 2% 76% 12% ND-8610 523 AGGACCCUAGACCUCUGCATsT 524 UGCAGAGGUCUAGGGUCCUTsT 61%

% 73% 10% ND-8611 525 CCACCGCUCCUACCGAGAGTsT 526 CUCUCGGUAGGAGCGGUGGTsT 21% 2% 29%

% ND-8612 527 UACCGAGAGCUCUUCGAGUTsT 528 ACUCGAAGAGCUCUCGGUATsT 13% 1% 22% 3% ND-8613 529 AACAUCCUGUCGAGGCUGCTsT 530 GCAGCCUCGACAGGAUGUUTsT 57% 2% 70% 4% ND-8614 531 GAACCUUUACCCUUCAAAGTsT 532 CUUUGAAGGGUAAAGGUUCTsT 13% 3% 16% 3% ND-8615 533 GGUUCCGAAGCCGAUACUGTsT 534 CAGUAUCGGCUUCGGAACCTsT 18% 1% 24% 2% ND-8616 535 AAGCCGAUACUGGUCUCCATsT 536 UGGAGACCAGUAUCGGCUUTsT 19% 1% 25% 3% ND-8617 537 UCUAGCCCAUCAUCCUGCUTsT 538 AGCAGGAUGAUGGGCUAGATsT 93% 3% 101%  3% ND-8618 539 CGGCGCCAUCCGCCUGGUGTsT 540 CACCAGGCGGAUGGCGCCGTsT 85% 4% 99% 4% ND-8619 541 UUUUCGGAGAGUACUUCAGTsT 542 CUGAAGUACUCUCCGAAAATsT 63% 2% 77% 3% ND-8620 543 GAGAGUACUUCAGCUACCCTsT 544 GGGUAGCUGAAGUACUCUCTsT 26% 1% 30% 4% ND-8621 545 GACGCUCUUUGACCUGUACTsT 546 GUACAGGUCAAAGAGCGUCTsT 17% 2% 19% 3% ND-8622 547 UGUGUAUUCACUCCUGCUUTsT 548 AAGCAGGAGUGAAUACACATsT 49% 3%

8% 11%  ND-8623 549 AACAACAAGAGAAAUGGAGTsT 550 CUCCAUUUCUCUUGUUGUUTsT 74% 7% 70% 4% ND-8624 551 AUUGAAGGAUGUGCAGGGCTsT 552 GCCCUGCACAUCCUUCAAUTsT 85% 6% 87% 12%  ND-8625 553 UCUCAGAGCCGCCCAAACUTsT 554 AGUUUGGGCGGCUCUGAGATsT 53% 3% 51% 6% ND-8626 555 AAACACAACCAAGGGUACATsT 556 UGUACCCUUGGUUGUGUUUTsT 17% 2% 18% 2% ND-8627 557 UACCCGUGCCCUCACAGAGTsT 558 CUCUGUGAGGGCACGGGUATsT 58% 3% 55% 3% ND-8628 559 UAGCACACUAUAACAUCUGTsT 560 CAGAUGUUAUAGUGUGCUATsT 64% 3% 64% 1

%  ND-8629 561 GGUGUGUAUUCACUCCUGCTsT 562 GCAGGAGUGAAUACACACCTsT 25% 3% 23% 2% ND-8630 563 CAUGAUCAAGGAGUGUGGCTsT 564 GCCACACUCCUUGAUCAUGTsT 32% 2% 28% 2% ND-8631 565 ACUCACGAUGGCCCUCGGUTsT 566 ACCGAGGGCCAUCGUGAGUTsT 96% 1% 88% 4% ND-8632 567 GGAGCUUUGACAAGGAACUTsT 568 AGUUCCUUGUCAAAGCUCCTsT 14% 1% 14% 2% ND-8633 569 AUACCCGUGCCCUCACAGATsT 570 UCUGUGAGGGCACGGGUAUTsT 21% 2% 16% 1% ND-8634 571 GGAGUGGCCAAAGUCAACATsT 572 UGUUGACUUUGGCCACUCCTsT 21% 3% 16% 1% ND-8635 573 AACUACAAAACCAAUUCUGTsT 574 CAGAAUUGGUUUUGUAGUUTsT 49%

% 37% 3% ND-8636 575 UGCUGGAGUGUUGCUGUUGTsT 576 CAACAGCAACACUCCAGCATsT 27% 3% 21% 2% ND-8637 577 AGGUCUCCUGCAACCAGGCTsT 578 GCCUGGUUGCAGGAGACCUTsT 62% 8% 61% 4% ND-8638 579 CUUUGGCAUGAUGUACUGGTsT 580 CCAGUACAUCAUGCCAAAGTsT 66% 6% 52% 8% ND-8639 581 CAUCUGCACCCUCAAUCCCTsT 582 GGGAUUGAGGGUGCAGAUGTsT 50% 7% 40% 4% ND-8640 583 CGACUGCACCAAGAAUGGCTsT 584 GCCAUUCUUGGUGCAGUCGTsT 67% 6% 54% 5% ND-8641 585 AAAACACAACCAAGGGUACTsT 586 GUACCCUUGGUUGUGUUUUTsT 14% 2% 14% 1% ND-8642 587 CAUCUGCUGGAGUGUUGCUTsT 588 AGCAACACUCCAGCAGAUGTsT 13% 2% 13% 1% ND-8643 589 CCUACAUCUUCUAUCCGCGTsT 590 CGCGGAUAGAAGAUGUAGGTsT 15% 4% 13% 0% ND-8644 591 GCCUACAUCUUCUACUCGCTsT 592 GCGGAUAGAAGAUGUAGGCTsT 14% 3% 11% 1% ND-8645 593 GAGUGGUACCGCUUCCACUTsT 594 AGUGGAAGCGGUACCACUCTsT 16% 0% 20% 1% ND-8646 595 GGUACCGCUUCCACUACAUTsT 596 AUGUAGUGGAAGCGGUACCTsT 12% 0% 14% 1% ND-8647 597 GUGGUACCGCUUCCACUACTsT 598 GUAGUGGAAGCGGUACCACTsT 42% 4% 44% 3% ND-8648 599 GAAUUACUCUCACUUCCACTsT 600 GUGGAAGUGAGAGUAAUUCTsT 10% 1% 11% 3% ND-8649 601 AAUUACUCUCACUUCCACCTsT 602 GGUGGAAGUGAGAGUAAUUTsT 105%  10%  102%  8% ND-8650 603 UACUCUCACUUCCACCACCTsT 604 GGUGGUGGAAGUGAGAGUATsT 55% 6% 54% 8% ND-8651 605 AGUGGUACCGCUUCCACUATsT 606 UAGUGGAAGCGGUACCACUTsT 67% 6% 69% 12%  ND-8652 607 GGGCAACUUCAUCUUCGCCTsT 608 GGCGAAGAUGAAGUUGCCCTsT 47% 12%  36% 7%

indicates data missing or illegible when filed

TABLE 1B Selected siRNAs in extended screening set (“human-only” siRNAs). A further 344 iRNA sequences were identified and were designed to be fully complementary to the human  alpha-ENaC sequences, according to the design criteria described in the examples section. All siRNAs listed in this screening set were modified only with a phosphorothioate linkage at the  3′-end between nucleotides 20 and 21 of each strand. The percentage residual expression of  alpha-ENaC in single-dose transfection assay is shown (refer to examples section for methods  used). 1st 2nd screen screen single single dose dose @ 50 nM @ 50 nM in H441; in H441; Duplex ID Seq ID Sense Seq ID Antissense  MV SD  MV SD ND-10445 609 CUGCGGCUAAGUCUCUUUUTsT 610 AAAAGAGACUUAGCCGCAGTsT 84% 8% ND-10446 611 AUCGCGACAGAACAAUUACTsT 612 GUAAUUGUUCUGUCGCGAUTsT 1

% 2% ND-10447 613 UCGCGACAGAACAAUUACATsT 614 UGUAAUUGUUCUGUCGCGATsT 18% 1% ND-10448 615 CCCGUUUAUGUAUGCUCCATsT 616 UGGAGCAUACAUAAACGGGTsT 41% 1% ND-10449 617 CCCGGGUAAGUAAAGGCAGTsT 618 CUGCCUUUACUUACCCGGGTsT 23% 1% ND-10450 619 GGUACCCGGAAAUUAAAGATsT 620 UCUUUAAUUUCCGGGUACCTsT 14% 2% ND-10451 621 GCUAUCGCGACAGAACAAUTsT 622 AUUGUUCUGUCGCGAUAGCTsT 24% 2% ND-10452 623 UAUCGCGACAGAACAAUUATsT 624 UAAUUGUUCUGUCGCGAUATsT 12% 1% ND-10453 625 UGCGGCUAAGUCUCUUUUUTsT 626 AAAAAGAGACUUAGCCGCATsT 46% 3% ND-10454 627 GGCGAUUAUGGCGACUGCATsT 628 UGCAGUCGCCAUAAUCGCCTsT 14% 0% ND-10455 629 AUGUCUAGCCCAUCAUCCUTsT 630 AGGAUGAUGGGCUAGACAUTsT 12% 2% ND-10456 631 CUACAGGUACCCGGAAAUUTsT 632 AAUUUCCGGGUACCUGUAGTsT 28% 2% ND-10457 633 CUACAGGUACCCGGAAAUUTsT 634 CACGCUACGGGCUCGACGGTsT 27% 3% ND-10458 635 CGCGACAGAACAAUUACACTsT 636 GUGUAAUUGUUCUGUCGCGTsT 39% 7% ND-10459 637 AGGUACCCGGAAAUUAAAGTsT 638 CUUUAAUUUCCGGGUACCUTsT 30% 3% ND-10460 639 CAUGCACGGGUUUCCUGCCTsT 640 GGCAGGAAACCCGUGCAUGTsT 9

% 6% ND-10461 641 AGCUUGCGGGACAACAACCTsT 642 GGUUGUUGUCCCGCAAGCUTsT 94% 8% ND-10462 643 ACUGCGGCUAAGUCUCUUUTsT 644 AAAGAGACUUAGCCGCAGUTsT 13% 2% ND-10463 645 CAUCCCUUAGAACCCUGCUTsT 646 AGCAGGGUUCUAAGGGAUGTsT 18% 1% ND-10464 647 ACCCGGGUAAGUAAAGGCATsT 648 UGCCUUUACUUACCCGGGUTsT 41% 1% ND-10465 649 GAUUAUGGCGACUGCACCATsT 650 UGGUGCAGUCGCCAUAAUCTsT 23% 1% ND-10466 651 CUCGGACAAGCUCGUCUUCTsT 652 GAAGACGAGCUUGUCCGAGTsT 14% 2% ND-10467 653 GCGAUUAUGGCGACUGCACTsT 654 GUGCAGUCGCCAUAAUCGCTsT 24% 2% ND-10468 655 AAUUACACCGUCAACAACATsT 656 UGUUGUUGACGGUGUAAUUTsT 12% 1% ND-10469 657 AACUGCCGUUGAUGUGUGGTsT 658 CCACACAUCAACGGCAGUUTsT 46% 3% ND-10470 659 AACUGCGGCUAAGUCUCUUTsT 660 AAGAGACUUAGCCGCAGUUTsT 14% 0% ND-10471 661 CCGCUGAUAACCAGGACAATsT 662 UUGUCCUGGUUAUCAGCGGTsT 12% 2% ND-10472 663 AAGGGUACACGCAGGCAUGTsT 664 CAUGCCUGCGUGUACCCUUTsT 28% 2% ND-10473 665 CCGGGUAAGUAAAGGCAGATsT 666 UCUGCCUUUACUUACCCGGTsT 27% 3% ND-10474 667 CCCAUACCAGGUCUCAUGGTsT 668 CCAUGAGACCUGGUAUGGGTsT 39% 7% ND-10475 669 AUUAUGGCGACUGCACCAATsT 670 UUGGUGCAGUCGCCAUAAUTsT 30% 3% ND-10476 671 AUGCACGGGUUUCCUGCCCTsT 672 GGGCAGGAAACCCGUGCAUTsT 95% 6% ND-10477 673 CUAGCCCUCCACAGUCCACTsT 674 GUGGACUGUGGAGGGCUAGTsT 43% 7% ND-10478 675 CAGGUACCCGGAAAUUAAATsT 676 UUUAAUUUCCGGGUACCUGTsT 11% 1% ND-10479 677 AAUACAGCUCCUUCACCACTsT 678 GUGGUGAAGGAGCUGUAUUTsT 30% 3% ND-10480 679 CACGGGUUUCCUGCCCAGCTsT 680 GCUGGGCAGGAAACCCGUGTsT 19% 1% ND-10481 681 GGACUGAAUCUUGCCCGUUTsT 682 AACGGGCAAGAUUCAGUCCTsT 14% 2% ND-10482 683 CGUUUAUGUAUGCUCCAUGTsT 684 CAUGGAGCAUACAUAAACGTsT 15% 1% ND-10483 685 GGGUACUGCUACUAUAAGCTsT 686 GCUUAUAGUAGCAGUACCCTsT 11% 0% ND-10484 687 UCGGUGUUGUCUGUGGUGGTsT 688 CCACCACAGACAACACCGATsT 65% 5% ND-10485 689 AAACUGCCGUUGAUGUGUGTsT 690 CACACAUCAACGGCAGUUUTsT 73% 6% ND-10486 691 GCGAAACUUGGAGCUUUGATsT 692 UCAAAGCUCCAAGUUUCGCTsT  8% 1% ND-10487 693 GGCCCGUCGAGCCCGUAGCTsT 694 GCUACGGGCUCGACGGGCCTsT 26% 3% ND-10488 695 GCGACAGAACAAUUACACCTsT 696 GGUGUAAUUGUUCUGUCGCTsT 10% 2% ND-10489 697 GCGACGGCUUAAGCCAGCCTsT 698 GGCUGGCUUAAGCCGUCGCTsT 50% 1% ND-10490 699 GACCCGGGUAAGUAAAGGCTsT 700 GCCUUUACUUACCCGGGUCTsT 74% 1% ND-10491 701 UUGAUCACUCCGCCUUCUCTsT 702 GAGAAGGCGGAGUGAUCAATsT 80% 7% ND-10492 703 UCUAGCCCUCCACAGUCCATsT 704 UGGACUGUGGAGGGCUAGATsT 69% 4% ND-10493 705 GUUUCACCAAGUGCCGGAATsT 706 UUCCGGCACUUGGUGAAACTsT 23% 3% ND-10494 707 CUCAACUCGGACAAGCUCGTsT 708 CGAGCUUGUCCGAGUUGAGTsT 45% 6% ND-10495 709 CAACUCGGACAAGCUCGUCTsT 710 GACGAGCUUGUCCGAGUUGTsT 23% 3% ND-10496 711 ACCCGGAAAUUAAAGAGGATsT 712 UCCUCUUUAAUUUCCGGGUTsT 13% 2% ND-10497 713 CCCGGAAAUUAAAGAGGAGTsT 714 CUCCUCUUUAAUUUCCGGGTsT 19% 1% ND-10498 715 CACCACUCUCGUGGCCGGCTsT 716 GCCGGCCACGAGAGUGGUGTsT 94% 11%  ND-10499 717 CGUCGAGCCCGUAGCGUGGTsT 718 CCACGCUACGGGCUCGACGTsT 13% 1% ND-10500 719 GCUUGCGGGACAACAACCCTsT 720 GGGUUGUUGUCCCGCAAGCTsT 49% 2% ND-10501 721 GAAUCAACAACGGUCUGUCTsT 722 GACAGACCGUUGUUGAUUCTsT 18% 2% ND-10502 723 GGGCGAUUAUGGCGACUGCTsT 724 GCAGUCGCCAUAAUCGCCCTsT  8% 1% ND-10503 725 CGAUUAUGGCGACUGCACCTsT 726 GGUGCAGUCGCCAUAAUCGTsT 17% 1% ND-10504 727 UCUGCUGGUUACUCACGAUTsT 728 AUCGUGAGUAACCAGCAGATsT 38% 4% ND-10505 729 CUAUCGCGACAGAACAAUUTsT 730 AAUUGUUCUGUCGCGAUAGTsT  9% 1% ND-10506 731 CAAUUACACCGUCAACAACTsT 732 GUUGUUGACGGUGUAAUUGTsT 11% 1% ND-10507 733 ACCGUCAACAACAAGAGAATsT 734 UUCUCUUGUUGUUGACGGUTsT  9% 1% ND-10508 735 CUCCUCGGUGUUGUCUGUGTsT 736 CACAGACAACACCGAGGAGTsT 78% 5% ND-10509 737 GGAGGUAGCCUCCACCCUGTsT 738 CAGGGUGGAGGCUACCUCCTsT 18% 1% ND-10510 739 GGAGAGGUUUCUCACACCATsT 740 UGGUGUGAGAAACCUCUCCTsT 13% 1% ND-10511 741 CUGCCGUUGAUGUGUGGAGTsT 742 CUCCACACAUCAACGGCAGTsT 19% 2% ND-10512 743 UGCCGUUGAUGUGUGGAGGTsT 744 CCUCCACACAUCAACGGCATsT 82% 4% ND-10513 745 AGAUGGGUAAGGGCUCAGGTsT 746 CCUGAGCCCUUACCCAUCUTsT 24% 1% ND-10514 747 AGAACAGUAGCUGAUGAAGTsT 748 CUUCAUCAGCUACUGUUCUTsT 15% 0% ND-10515 749 GCGGCUAAGUCUCUUUUUCTsT 750 GAAAAAGAGACUUAGCCGCTsT 13% 1% ND-10516 751 CCUAAGAAACCGCUGAUAATsT 752 UUAUCAGCGGUUUCUUAGGTsT  6% 0% ND-10517 753 GAAACCGCUGAUAACCAGGTsT 754 CCUGGUUAUCAGCGGUUUCTsT 13% 0% ND-10518 755 AACCGCUGAUAACCAGGACTsT 756 GUCCUGGUUAUCAGCGGUUTsT 42% 2% ND-10519 757 ACCGCUGAUAACCAGGACATsT 758 UGUCCUGGUUAUCAGCGGUTsT 11% 1% ND-10520 759 CCAAGGGUACACGCAGGCATsT 760 UGCCUGCGUGUACCCUUGGTsT 19% 1% ND-10521 761 CAAGGGUACACGCAGGCAUTsT 762 AUGCCUGCGUGUACCCUUGTsT 12% 0% ND-10522 763 AGGGUACACGCAGGCAUGCTsT 764 GCAUGCCUGCGUGUACCCUTsT 23% 1% ND-10523 765 GUACACGCAGGCAUGCACGTsT 766 CGUGCAUGCCUGCGUGUACTsT 27% 1% ND-10524 767 AGGCAUGCACGGGUUUCCUTsT 768 AGGAAACCCGUGCAUGCCUTsT 14% 0% ND-10525 769 GGCAUGCACGGGUUUCCUGTsT 770 CAGGAAACCCGUGCAUGCCTsT 18% 3% ND-10526 771 ACGGGUUUCCUGCCCAGCGTsT 772 CGCUGGGCAGGAAACCCGUTsT 30% 1% ND-10527 773 GAGCAGACCCGGGUAAGUATsT 774 UACUUACCCGGGUCUGCUCTsT 24% 2% ND-10528 775 AGCAGACCCGGGUAAGUAATsT 776 UUACUUACCCGGGUCUGCUTsT 24% 2% ND-10529 777 GGGUAAGUAAAGGCAGACCTsT 778 GGUCUGCCUUUACUUACCCTsT 39% 3% ND-10530 779 AGCCUCAUACCCGUGCCCUTsT 780 AGGGCACGGGUAUGAGGCUTsT 82% 5% ND-10531 781 GUGAACGCUUCUGCCACAUTsT 782 AUGUGGCAGAAGCGUUCACTsT 13% 1% ND-10532 783 AAAUUGAUCACUCCGCCUUTsT 784 AAGGCGGAGUGAUCAAUUUTsT 18% 2% ND-10533 785 AAUUGAUCACUCCGCCUUCTsT 786 GAAGGCGGAGUGAUCAAUUTsT 19% 0% ND-10534 787 GCCUUGCGGUCAGGGACUGTsT 788 CAGUCCCUGACCGCAAGGCTsT 12% 1% ND-10535 789 CUUGCGGUCAGGGACUGAATsT 790 UUCAGUCCCUGACCGCAAGTsT 11% 0% ND-10536 791 UUGCGGUCAGGGACUGAAUTsT 792 AUUCAGUCCCUGACCGCAATsT 12% 0% ND-10537 793 AUGUAUGCUCCAUGUCUAGTsT 794 CUAGACAUGGAGCAUACAUTsT 21% 1% ND-10538 795 AGCAAGUAGGCAGGAGCUCTsT 796 GAGCUCCUGCCUACUUGCUTsT 19% 1% ND-10539 797 CAGCCCAUACCAGGUCUCATsT 798 UGAGACCUGGUAUGGGCUGTsT 27% 2% ND-10540 799 CAGCCGUCGCGACCUGCGGTsT 800 CCGCAGGUCGCGACGGCUGTsT 44% 4% ND-10541 801 GGGCCCGUCGAGCCCGUAGTsT 802 CUACGGGCUCGACGGGCCCTsT 71% 6% ND-10542 803 CGUAGCGUGGCCUCCAGCUTsT 804 AGCUGGAGGCCACGCUACGTsT 84% 9% ND-10543 805 GGUGAGGGAGUGGUACCGCTsT 806 GCGGUACCACUCCCUCACCTsT 108%  8% ND-10544 807 AAAGUACACACAGCAGGUGTsT 808 CACCUGCUGUGUGUACUUUTsT 140%  7% ND-10545 809 CCAGGUUGACUUCUCCUCATsT 810 UGAGGAGAAGUCAACCUGGTsT 18% 2% ND-10546 811 UGUUUCACCAAGUGCCGGATsT 812 UCCGGCACUUGGUGAAACATsT 31% 2% ND-10547 813 UGCUGGUUACUCACGAUGGTsT 814 CCAUCGUGAGUAACCAGCATsT 144%  10%  ND-10548 815 UCCUCGGUGUUGUCUGUGGTsT 816 CCACAGACAACACCGAGGATsT 106%  14%  ND-10549 817 AGGUAGCCUCCACCCUGGCTsT 818 GCCAGGGUGGAGGCUACCUTsT 74% 15%  ND-10550 819 GCCGUUGAUGUGUGGAGGGTsT 820 CCCUCCACACAUCAACGGCTsT 26% 4% ND-10551 821 GAUGGGUAAGGGCUCAGGATsT 822 UCCUGAGCCCUUACCCAUCTsT 22% 1% ND-10552 823 CCCAACUGCGGCUAAGUCUTsT 824 AGACUUAGCCGCAGUUGGGTsT 18% 2% ND-10553 825 CCAAGCGAAACUUGGAGCUTsT 826 AGCUCCAAGUUUCGCUUGGTsT 16% 1% ND-10554 827 GGGUACACGCAGGCAUGCATsT 828 UGCAUGCCUGCGUGUACCCTsT 19% 2% ND-10555 829 UGCACGGGUUUCCUGCCCATsT 830 UGGGCAGGAAACCCGUGCATsT 28% 2% ND-10556 831 CUCCUCUAGCCUCAUACCCTsT 832 GGGUAUGAGGCUAGAGGAGTsT 109%  8% ND-10557 833 UCCUCUAGCCUCAUACCCGTsT 834 CGGGUAUGAGGCUAGAGGATsT 117%  7% ND-10558 835 UCUAGCCUCAUACCCGUGCTsT 836 GCACGGGUAUGAGGCUAGATsT 128%  9% ND-10559 837 UUCAUACCUCUACAUGUCUTsT 838 AGACAUGUAGAGGUAUGAATsT 52% 4% ND-10560 839 UCUACAUGUCUGCUUGAGATsT 840 UCUCAAGCAGACAUGUAGATsT 15% 2% ND-10561 841 AUAUUUCCUCAGCCUGAAATsT 842 UUUCAGGCUGAGGAAAUAUTsT 15% 2% ND-10562 843 AACUCCUAUGCAUCCCUUATsT 844 UAAGGGAUGCAUAGGAGUUTsT 14% 1% ND-10563 845 GCAUCCCUUAGAACCCUGCTsT 846 GCAGGGUUCUAAGGGAUGCTsT 20% 1% ND-10564 847 UGAUCACUCCGCCUUCUCCTsT 848 GGAGAAGGCGGAGUGAUCATsT 67% 7% ND-10565 849 UGUAAGUGCCUUGCGGUCATsT 850 UGACCGCAAGGCACUUACATsT 17% 2% ND-10566 851 CCUUGCGGUCAGGGACUGATsT 852 UCAGUCCCUGACCGCAAGGTsT 14% 1% ND-10567 853 AAUCUUGCCCGUUUAUGUATsT 854 UACAUAAACGGGCAAGAUUTsT 13% 2% ND-10568 855 CCGUUUAUGUAUGCUCCAUTsT 856 AUGGAGCAUACAUAAACGGTsT 19% 6% ND-10569 857 UGUAUGCUCCAUGUCUAGCTsT 858 GCUAGACAUGGAGCAUACATsT 87% 13%  ND-10570 859 CAUGUCUAGCCCAUCAUCCTsT 860 GGAUGAUGGGCUAGACAUGTsT 33% 4% ND-10571 861 AGUAGGCAGGAGCUCAAUATsT 862 UAUUGAGCUCCUGCCUACUTsT 11% 1% ND-10572 863 CCUACAGGUACCCGGAAAUTsT 864 AUUUCCGGGUACCUGUAGGTsT 22% 3% ND-10573 865 CCCGUCGAGCCCGUAGCGUTsT 866 ACGCUACGGGCUCGACGGGTsT 23% 1% ND-10574 867 GCGGUGAGGGAGUGGUACCTsT 868 GGUACCACUCCCUCACCGCTsT 30% 1% ND-10575 869 UUAUGGCGACUGCACCAAGTsT 870 CUUGGUGCAGUCGCCAUAATsT 77% 6% ND-10576 871 CUAUAAGCUCCAGGUUGACTsT 872 GUCAACCUGGAGCUUAUAGTsT 11% 1% ND-10577 873 UAUAAGCUCCAGGUUGACUTsT 874 AGUCAACCUGGAGCUUAUATsT 42% 8% ND-10578 875 AGGUUGACUUCUCCUCAGATsT 876 UCUGAGGAGAAGUCAACCUTsT 13% 3% ND-10579 877 CUGGGCUGUUUCACCAAGUTsT 878 ACUUGGUGAAACAGCCCAGTsT 19% 6% ND-10580 879 AACAAUUACACCGUCAACATsT 880 UGUUGACGGUGUAAUUGUUTsT 13% 1% ND-10581 881 UGGGUAAGGGCUCAGGAAGTsT 882 CUUCCUGAGCCCUUACCCATsT 20% 3% ND-10582 883 GGGUAAGGGCUCAGGAAGUTsT 884 ACUUCCUGAGCCCUUACCCTsT 22% 3% ND-10583 885 CACCCAACUGCGGCUAAGUTsT 886 ACUUAGCCGCAGUUGGGUGTsT 22% 10%  ND-10584 887 ACCCAACUGCGGCUAAGUCTsT 888 GACUUAGCCGCAGUUGGGUTsT 22% 5% ND-10585 889 CCAACUGCGGCUAAGUCUCTsT 890 GAGACUUAGCCGCAGUUGGTsT 14% 2% ND-10586 891 CUUGGAUCAGCCAAGCGAATsT 892 UUCGCUUGGCUGAUCCAAGTsT 15% 1% ND-10587 893 GCCAAGCGAAACUUGGAGCTsT 894 GCUCCAAGUUUCGCUUGGCTsT 17% 2% ND-10588 895 UCCUAAGAAACCGCUGAUATsT 896 UAUCAGCGGUUUCUUAGGATsT 11% 2% ND-10589 897 GCAUGCACGGGUUUCCUGCTsT 898 GCAGGAAACCCGUGCAUGCTsT 24% 8% ND-10590 899 UGUUACUUAGGCAAUUCCCTsT 900 GGGAAUUGCCUAAGUAACATsT 48% 10%  ND-10591 901 CUAGGGCUAGAGCAGACCCTsT 902 GGGUCUGCUCUAGCCCUAGTsT 58% 10%  ND-10592 903 CUCUAGCCUCAUACCCGUGTsT 904 CACGGGUAUGAGGCUAGAGTsT 34% 5% ND-10593 905 UUAGAACCCUGCUCAGACATsT 906 UGUCUGAGCAGGGUUCUAATsT 14% 1% ND-10594 907 UGUGAACGCUUCUGCCACATsT 908 UGUGGCAGAAGCGUUCACATsT 15% 0% ND-10595 909 AUUGAUCACUCCGCCUUCUTsT 910 AGAAGGCGGAGUGAUCAAUTsT 43% 1% ND-10596 911 UCACUCCGCCUUCUCCUGGTsT 912 CCAGGAGAAGGCGGAGUGATsT 90% 5% ND-10597 913 GCGGUCAGGGACUGAAUCUTsT 914 AGAUUCAGUCCCUGACCGCTsT 11% 0% ND-10598 915 GGUCAGGGACUGAAUCUUGTsT 916 CAAGAUUCAGUCCCUGACCTsT 13% 1% ND-10599 917 GUAUGCUCCAUGUCUAGCCTsT 918 GGCUAGACAUGGAGCAUACTsT 28% 3% ND-10600 919 CCAUGUCUAGCCCAUCAUCTsT 920 GAUGAUGGGCUAGACAUGGTsT 12% 1% ND-10601 921 GAUCGAGUUCCACCGCUCCTsT 922 GGAGCGGUGGAACUCGAUCTsT 17% 1% ND-10602 923 GGACUCUAGCCCUCCACAGTsT 924 CUGUGGAGGGCUAGAGUCCTsT 41% 4% ND-10603 925 UCACCACUCUCGUGGCCGGTsT 926 CCGGCCACGAGAGUGGUGATsT 83% 3% ND-10604 927 CAGCUUGCGGGACAACAACTsT 928 GUUGUUGUCCCGCAAGCUGTsT 21% 1% ND-10605 929 CAUCUUCUAUCCGCGGCCCTsT 930 GGGCCGCGGAUAGAAGAUGTsT 26% 2% ND-10606 931 AUAAGCUCCAGGUUGACUUTsT 932 AAGUCAACCUGGAGCUUAUTsT 15% 1% ND-10607 933 CUGCUGGUUACUCACGAUGTsT 934 CAUCGUGAGUAACCAGCAGTsT 85% 8% ND-10608 935 GAACAAUUACACCGUCAACTsT 936 GUUGACGGUGUAAUUGUUCTsT 13% 1% ND-10609 937 AUUACACCGUCAACAACAATsT 938 UUGUUGUUGACGGUGUAAUTsT 12% 0% ND-10610 939 CUGUGGUUCGGCUCCUCGGTsT 940 CCGAGGAGCCGAACCACAGTsT 53% 2% ND-10611 941 GAAGUGCCUUGGCUCCAGCTsT 942 GCUGGAGCCAAGGCACUUCTsT 24% 3% ND-10612 943 GAUCAGCCAAGCGAAACUUTsT 944 AAGUUUCGCUUGGCUGAUCTsT 12% 0% ND-10613 945 AGAAACCGCUGAUAACCAGTsT 946 CUGGUUAUCAGCGGUUUCUTsT 12% 1% ND-10614 947 UGAUAACCAGGACAAAACATsT 948 UGUUUUGUCCUGGUUAUCATsT  7% 1% ND-10615 949 CACGCAGGCAUGCACGGGUTsT 950 ACCCGUGCAUGCCUGCGUGTsT 12% 0% ND-10616 951 GCUCUCCAGUAGCACAGAUTsT 952 AUCUGUGCUACUGGAGAGCTsT  9% 1% ND-10617 953 CAGACCCGGGUAAGUAAAGTsT 954 CUUUACUUACCCGGGUCUGTsT 55% 3% ND-10618 955 AGACCCGGGUAAGUAAAGGTsT 956 CCUUUACUUACCCGGGUCUTsT 72% 8% ND-10619 957 AUCACUCCGCCUUCUCCUGTsT 958 CAGGAGAAGGCGGAGUGAUTsT 63% 6% ND-10620 959 CACUCCGCCUUCUCCUGGGTsT 960 CCCAGGAGAAGGCGGAGUGTsT 28% 1% ND-10621 961 AACUAGACUGUAAGUGCCUTsT 962 AGGCACUUACAGUCUAGUUTsT 23% 1% ND-10622 963 UAUGCUCCAUGUCUAGCCCTsT 964 GGGCUAGACAUGGAGCAUATsT 98% 2% ND-10623 965 CCCGAUGUAUGGAAACUGCTsT 966 GCAGUUUCCAUACAUCGGGTsT 11% 1% ND-10624 967 GUACUGCUACUAUAAGCUCTsT 968 GAGCUUAUAGUAGCAGUACTsT 19% 1% ND-10625 969 AGCGUGACCAGCUACCAGCTsT 970 GCUGGUAGCUGGUCACGCUTsT 49% 2% ND-10626 971 ACAAUUACACCGUCAACAATsT 972 UUGUUGACGGUGUAAUUGUTsT  8% 0% ND-10627 973 AUGCUCCUCUGGUGGGAGGTsT 974 CCUCCCACCAGAGGAGCAUTsT 76% 5% ND-10628 975 AACAGUAGCUGAUGAAGCUTsT 976 AGCUUCAUCAGCUACUGUUTsT 22% 1% ND-10629 977 CUGACUCCCGAGGGCUAGGTsT 978 CCUAGCCCUCGGGAGUCAGTsT 34% 2% ND-10630 979 GUGCAACCAGAACAAAUCGTsT 980 CGAUUUGUUCUGGUUGCACTsT 10% 1% ND-10631 981 UGCAACCAGAACAAAUCGGTsT 982 CCGAUUUGUUCUGGUUGCATsT 48% 4% ND-10632 983 CUUCAAAGUACACACAGCATsT 984 UGCUGUGUGUACUUUGAAGTsT 20% 1% ND-10633 985 CAGCGUGACCAGCUACCAGTsT 986 CUGGUAGCUGGUCACGCUGTsT 35% 1% ND-10634 987 AGAACAAUUACACCGUCAATsT 988 UUGACGGUGUAAUUGUUCUTsT 14% 0% ND-10635 989 GAUAACCAGGACAAAACACTsT 990 GUGUUUUGUCCUGGUUAUCTsT 11% 1% ND-10636 991 ACAACCAAGGGUACACGCATsT 992 UGCGUGUACCCUUGGUUGUTsT 17% 1% ND-10637 993 CCCAGCGACGGCUUAAGCCTsT 994 GGCUUAAGCCGUCGCUGGGTsT 27% 2% ND-10638 995 CUCCCGAGGGCUAGGGCUATsT 996 UAGCCCUAGCCCUCGGGAGTsT 23% 1% ND-10639 997 UAGAACCCUGCUCAGACACTsT 998 GUGUCUGAGCAGGGUUCUATsT 35% 2% ND-10640 999 CCUGGGCUGUUUCACCAAGTsT 1000 CUUGGUGAAACAGCCCAGGTsT 14% 1% ND-10641 1001 GGAUCAGCCAAGCGAAACUTsT 1002 AGUUUCGCUUGGCUGAUCCTsT 16% 3% ND-10642 1003 AAGAAACCGCUGAUAACCATsT 1004 UGGUUAUCAGCGGUUUCUUTsT 17% 1% ND-10643 1005 ACCAAGGGUACACGCAGGCTsT 1006 GCCUGCGUGUACCCUUGGUTsT 37% 4% ND-10644 1007 GUAGCACAGAUGUCUGCUCTsT 1008 GAGCAGACAUCUGUGCUACTsT 13% 3% ND-10645 1009 UUUCAUACCUCUACAUGUCTsT 1010 GACAUGUAGAGGUAUGAAATsT 88% 8% ND-10646 1011 CCAACCAUCUGCCAGAGAATsT 1012 UUCUCUGGCAGAUGGUUGGTsT 16% 2% ND-10647 1013 GUCAGGGACUGAAUCUUGCTsT 1014 GCAAGAUUCAGUCCCUGACTsT 16% 3% ND-10648 1015 AGCAUGAUCAAGGAGUGUGTsT 1016 CACACUCCUUGAUCAUGCUTsT 50% 7% ND-10649 1017 GCAGCGUGACCAGCUACCATsT 1018 UGGUAGCUGGUCACGCUGCTsT 40% 6% ND-10650 1019 CAGCUCUCUGCUGGUUACUTsT 1020 AGUAACCAGCAGAGAGCUGTsT 56% 5% ND-10651 1021 GUUCGGCUCCUCGGUGUUGTsT 1022 CAACACCGAGGAGCCGAACTsT 68% 5% ND-10652 1023 GCAGAUGCUCCUCUGGUGGTsT 1024 CCACCAGAGGAGCAUCUGCTsT 26% 5% ND-10653 1025 AGGAAGUUGCUCCAAGAACTsT 1026 GUUCUUGGAGCAACUUCCUTsT 18% 2% ND-10654 1027 AACGCUUCUGCCACAUCUUTsT 1028 AAGAUGUGGCAGAAGCGUUTsT 18% 1% ND-10655 1029 CACCUGGGCUGUUUCACCATsT 1030 UGGUGAAACAGCCCAGGUGTsT 17% 2% ND-10656 1031 AAGCCAUGCAGCGUGACCATsT 1032 UGGUCACGCUGCAUGGCUUTsT 27% 3% ND-10657 1033 CGAGGGCUAGGGCUAGAGCTsT 1034 GCUCUAGCCCUAGCCCUCGTsT 30% 1% ND-10658 1035 GGAAACCCUGGACAGACUUTsT 1036 AAGUCUGUCCAGGGUUUCCTsT 14% 1% ND-10659 1037 GUAGCUGAUGAAGCUGCCCTsT 1038 GGGCAGCUUCAUCAGCUACTsT 19% 1% ND-10660 1039 UCUUUUUCCCUUGGAUCAGTsT 1040 CUGAUCCAAGGGAAAAAGATsT 88% 4% ND-10661 1041 CUCCAGUAGCACAGAUGUCTsT 1042 GACAUCUGUGCUACUGGAGTsT 10% 1% ND-10662 1043 CCAAAAUUGAUCACUCCGCTsT 1044 GCGGAGUGAUCAAUUUUGGTsT 25% 3% ND-10663 1045 CAGACCACCUGGGCUGUUUTsT 1046 AAACAGCCCAGGUGGUCUGTsT 24% 2% ND-10664 1047 CCCUUCCCAACUAGACUGUTsT 1048 ACAGUCUAGUUGGGAAGGGTsT 15% 2% ND-10665 1049 CGCAGCCGUCGCGACCUGCTsT 1050 GCAGGUCGCGACGGCUGCGTsT 45% 2% ND-10666 1051 UUCUCACACCAAGGCAGAUTsT 1052 AUCUGCCUUGGUGUGAGAATsT 25% 2% ND-10667 1053 CACCACCAUCCACGGCGCCTsT 1054 GGCGCCGUGGAUGGUGGUGTsT 35% 3% ND-10668 1055 CCAUUACUUUUGUGAACGCTsT 1056 GCGUUCACAAAAGUAAUGGTsT 19% 4% ND-10669 1057 CCAAGAACAGUAGCUGAUGTsT 1058 CAUCAGCUACUGUUCUUGGTsT 23% 4% 16% 2% ND-10670 1059 AGGAGAGGUUUCUCACACCTsT 1060 GGUGUGAGAAACCUCUCCUTsT 18% 3% 17% 1% ND-10671 1061 AUCAUCCUGCUUGGAGCAATsT 1062 UUGCUCCAAGCAGGAUGAUTsT 33% 3% 24% 2% ND-10672 1063 GCAUCACAGAGCAGACGCUTsT 1064 AGCGUCUGCUCUGUGAUGCTsT 29% 2% 27% 4% ND-10673 1065 AGGAGGUAGCCUCCACCCUTsT 1066 AGGGUGGAGGCUACCUCCUTsT 63% 3% 61% 3% ND-10674 1067 ACAACCGCAUGAAGACGGCTsT 1068 GCCGUCUUCAUGCGGUUGUTsT 94% 2% ND-10675 1069 GCAUGAAGACGGCCUUCUGTsT 1070 CAGAAGGCCGUCUUCAUGCTsT 20% 2% 18% 1% ND-10676 1071 GUCACGAUGGUCACCCUCCTsT 1072 GGAGGGUGACCAUCGUCAGTsT 66% 5% 60% 4% ND-10677 1073 CCCUGCUCAGACACCAUUATsT 1074 UAAUGGUGUCUGAGCAGGGTsT 15% 3% 18% 1% ND-10678 1075 UCACGAUGGUCACCCUCCUTsT 1076 AGGAGGGUGACCAUCGUGATsT 80% 6% 70% 4% ND-10679 1077 UCAACCUCAACUCGGACAATsT 1078 UUGUCCGAGUUGAGGUUGATsT 20% 3% 21% 1% ND-10680 1079 UGACCAGCUACCAGCUCUCTsT 1080 GAGAGCUGGUAGCUGGUCATsT 88% 22%  77% 5% ND-10681 1081 GAUGGCCCUCGGUGACAUCTsT 1082 GAUGUCACCGAGGGCCAUCTsT 88% 18%  60% 4% ND-10682 1083 GCUUUGACAAGGAACUUUCTsT 1084 GAAAGUUCCUUGUCAAAGCTsT 19% 7% 14% 2% ND-10683 1085 CGAUACUGGUCUCCAGGCCTsT 1086 GGCCUGGAGACCAGUAUCGTsT 27% 5% 27% 2% ND-10684 1087 UCUGGAUGUCUUCCAUGCCTsT 1088 GGCAUGGAAGACAUCCAGATsT 92% 13%  89% 3% ND-10685 1089 CAGGACCCUAGACCUCUGCTsT 1090 GCAGAGGUCUAGGGUCCUGTsT 58% 14%  50% 2% ND-10686 1091 GACCCUAGACCUCUGCAGCTsT 1092 GCUGCAGAGGUCUAGGGUCTsT 27% 2% ND-10687 1093 ACCCUAGACCUCUGCAGCCTsT 1094 GGCUGCAGAGGUCUAGGGUTsT 21% 1% ND-10688 1095 CAGCCCACGGCGGAGGAGGTsT 1096 CCUCCUCCGCCGUGGGCUGTsT 55% 4% ND-10689 1097 CUCUUCGAGUUCUUCUGCATsT 1098 UGCAGAAGAACUCGAAGAGTsT 13% 3% ND-10690 1099 UUGGCAUGAUGUACUGGCATsT 1100 UGCCAGUACAUCAUGCCAATsT 16% 2% ND-10691 1101 GGCAUGAUGUACUGGCAAUTsT 1102 AUUGCCAGUACAUCAUGCCTsT 13% 1% ND-10692 1103 UGUACUGGCAAUUCGGCCUTsT 1104 AGGCCGAAUUGCCAGUACATsT 45% 2% ND-10693 1105 ACUGGCAAUUCGGCCUGCUTsT 1106 AGCAGGCCGAAUUGCCAGUTsT 38% 3% ND-10694 1107 GGCAAUUCGGCCUGCUUUUTsT 1108 AAAAGCAGGCCGAAUUGCCTsT 10% 1% ND-10695 1109 CAAUUCGGCCUGCUUUUCGTsT 1110 CGAAAAGCAGGCCGAAUUGTsT 12% 1% ND-10696 1111 UCGGAGAGUACUUCAGCUATsT 1112 UAGCUGAAGUACUCUCCGATsT 12% 1% ND-10697 1113 CAACAUCCUGUCGAGGCUGTsT 1114 CAGCCUCGACAGGAUGUUGTsT 35% 7% ND-10698 1115 CAUCCUGUCGAGGCUGCCATsT 1116 UGGCAGCCUCGACAGGAUGTsT 26% 6% ND-10699 1117 UCCUGCAACCAGGCGAAUUTsT 1118 AAUUCGCCUGGUUGCAGGATsT 28% 6% ND-10700 1119 GGAAACUGCUAUACUUUCATsT 1120 UGAAAGUAUAGCAGUUUCCTsT  7% 2% ND-10701 1121 ACGGUCUGUCCCUGAUGCUTsT 1122 AGCAUCAGGGACAGACCGUTsT 28% 7% ND-10702 1123 GGUCUGUCCCUGAUGCUGCTsT 1124 GCAGCAUCAGGGACAGACCTsT 33% 2% ND-10703 1125 GGCCCGGGUAAUGGUGCACTsT 1126 GUGCACCAUUACCCGGGCCTsT 47% 10%  ND-10704 1127 CAGGAUGAACCUGCCUUUATsT 1128 UAAAGGCAGGUUCAUCCUGTsT 59% 2% ND-10705 1129 GAUGAACCUGCCUUUAUGGTsT 1130 CCAUAAAGGCAGGUUCAUCTsT 77% 7% ND-10706 1131 GGUGGCUUUAACUUGCGGCTsT 1132 GCCGCAAGUUAAAGCCACCTsT 47% 8% ND-10707 1133 GUGGCUUUAACUUGCGGCCTsT 1134 GGCCGCAAGUUAAAGCCACTsT 17% 2% ND-10708 1135 UUGCGGCCUGGCGUGGAGATsT 1136 UCUCCACGCCAGGCCGCAATsT 52% 3% ND-10709 1137 UGCGGCCUGGCGUGGAGACTsT 1138 GUCUCCACGCCAGGCCGCATsT 81% 4% ND-10710 1139 GCGGCCUGGCGUGGAGACCTsT 1140 GGUCUCCACGCCAGGCCGCTsT 57% 4% ND-10711 1141 CAGGUGUGUAUUCACUCCUTsT 1142 AGGAGUGAAUACACACCUGTsT 24% 2% ND-10712 1143 GUGUAUUCACUCCUGCUUCTsT 1144 GAAGCAGGAGUGAAUACACTsT 20% 1% ND-10713 1145 GGCCCUCGGUGACAUCCCATsT 1146 UGGGAUGUCACCGAGGGCCTsT 40% 3% ND-10714 1147 GAUGCUAUCGCGACAGAACTsT 1148 GUUCUGUCGCGAUAGCAUCTsT 24% 2% ND-10715 1149 ACUACAAAACCAAUUCUGATsT 1150 UCAGAAUUGGUUUUGUAGUTsT 19% 2% ND-10716 1151 CAAUUCUGAGUCUCCCUCUTsT 1152 AGAGGGAGACUCAGAAUUGTsT 35% 3% ND-10717 1153 CUCUGUCACGAUGGUCACCTsT 1154 GGUGACCAUCGUGACAGAGTsT 41% 4% ND-10718 1155 CUGCUCCGAAGGUUCCGAATsT 1156 UUCGGAACCUUCGGAGCAGTsT 16% 3% ND-10719 1157 AGGUUCCGAAGCCGAUACUTsT 1158 AGUAUCGGCUUCGGAACCUTsT 16% 2% ND-10720 1159 GUUCCGAAGCCGAUACUGGTsT 1160 CCAGUAUCGGCUUCGGAACTsT 21% 2% ND-10721 1161 CGAAGCCGAUACUGGUCUCTsT 1162 GAGACCAGUAUCGGCUUCGTsT 16% 1% ND-10722 1163 AAGAUUGAAGGAUGUGCAGTsT 1164 CUGCACAUCCUUCAAUCUUTsT 25% 2% ND-10723 1165 GAUUGAAGGAUGUGCAGGGTsT 1166 CCCUGCACAUCCUUCAAUCTsT 26% 1% ND-10724 1167 UGCCUCUGAACACUCUGGUTsT 1168 ACCAGAGUGUUCAGAGGCATsT 45% 3% ND-10725 1169 CCUCUGAACACUCUGGUUUTsT 1170 AAACCAGAGUGUUCAGAGGTsT 15% 2% ND-10726 1171 GACAAGGAACUUUCCUAAGTsT 1172 CUUAGGAAAGUUCCUUGUCTsT 105%  14%  ND-10727 1173 CAGGACAAAACACAACCAATsT 1174 UUGGUUGUGUUUUGUCCUGTsT 32% 5% ND-10728 1175 AACACAACCAAGGGUACACTsT 1176 GUGUACCCUUGGUUGUGUUTsT 60% 13%  ND-10729 1177 UUGAACUUGGGUGGGAAACTsT 1178 GUUUCCCACCCAAGUUCAATsT 23% 8% ND-10730 1179 UGAACUUGGGUGGGAAACCTsT 1180 GGUUUCCCACCCAAGUUCATsT 18% 4% ND-10731 1181 ACCCGUGCCCUCACAGAGCTsT 1182 GCUCUGUGAGGGCACGGGUTsT 19% 1% ND-10732 1183 ACUAUAACAUCUGCUGGAGTsT 1184 CUCCAGCAGAUGUUAUAGUTsT 17% 5% ND-10733 1185 AUCUGCUGGAGUGUUGCUGTsT 1186 CAGCAACACUCCAGCAGAUTsT 119%  20%  ND-10734 1187 CUGCUGGAGUGUUGCUGUUTsT 1188 AACAGCAACACUCCAGCAGTsT 58% 13%  ND-10735 1189 CUAGCCCAUCAUCCUGCUUTsT 1190 AAGCAGGAUGAUGGGCUAGTsT 20% 6% ND-10736 1191 CUCUGGAUGUCUUCCAUGCTsT 1192 GCAUGGAAGACAUCCAGAGTsT 28% 8% ND-10737 1193 AGCAGGACCCUAGACCUCUTsT 1194 AGAGGUCUAGGGUCCUGCUTsT 36% 2% ND-10738 1195 UUCGAGUUCUUCUGCAACATsT 1196 UGUUGCAGAAGAACUCGAATsT 13% 1% ND-10739 1197 CACCAUCCACGGCGCCAUCTsT 1198 GAUGGCGCCGUGGAUGGUGTsT 13% 2% ND-10740 1199 CCACGGCGCCAUCCGCCUGTsT 1200 CAGGCGGAUGGCGCCGUGGTsT 44% 4% ND-10741 1201 CAGCACAACCGCAUGAAGATsT 1202 UCUUCAUGCGGUUGUGCUGTsT 23% 3% ND-10742 1203 CCUUUGGCAUGAUGUACUGTsT 1204 CAGUACAUCAUGCCAAAGGTsT 12% 1% ND-10743 1205 AUCCUGUCGAGGCUGCCAGTsT 1206 CIGGCAGCCUCGACAGGAUTsT 14% 3% ND-10744 1207 UCUCCUGCAACCAGGCGAATsT 1208 UUCGCCUGGUUGCAGGAGATsT 12% 1% ND-10745 1209 UGCAACCAGGCGAAUUACUTsT 1210 AGUAAUUCGCCUGGUUGCATsT 45% 5% ND-10746 1211 ACCUCCAUCAGCAUGAGGATsT 1212 UCCUCAUGCUGAUGGAGGUTsT 82% 7% ND-10747 1213 GCGACUGCACCAAGAAUGGTsT 1214 CCAUUCUUGGUGCAGUCGCTsT 82% 19%  ND-10748 1215 ACCAAGAAUGGCAGUGAUGTsT 1216 CAUCACUGCCAUUCUUGGUTsT 54% 18%  ND-10749 1217 UGGUUACUCACGAUGGCCCTsT 1218 GGGCCAUCGUGAGUAACCATsT 45% 7% ND-10750 1219 AGAAAUGGAGUGGCCAAAGTsT 1220 CUUUGGCCACUCCAUUUCUTsT 11% 3% ND-10751 1221 GGAGCUGAACUACAAAACCTsT 1222 GGUUUUGUAGUUCAGCUCCTsT 15% 3% ND-10752 1223 CCUCUGUCACGAUGGUCACTsT 1224 GUGACCAUCGUGACAGAGGTsT 18% 5% ND-10753 1225 AGAUUGAAGGAUGUGCAGGTsT 1226 CCUGCACAUCCUUCAAUCUTsT 26% 3% ND-10754 1227 GAGCUUUGACAAGGAACUUTsT 1228 AAGUUCCUUGUCAAAGCUCTsT 14% 2% ND-10755 1229 CUUUGACAAGGAACUUUCCTsT 1230 GGAAAGUUCCUUGUCAAAGTsT 50% 8% ND-10756 1231 UCAGACACCAUUACUUUUGTsT 1232 CAAAAGUAAUGGUGUCUGATsT 32% 4% ND-10757 1233 AGCACACUAUAACAUCUGCTsT 1234 GCAGAUGUUAUAGUGUGCUTsT 11% 2% ND-10758 1235 GCACAACCGCAUGAAGACGTsT 1236 CGUCUUCAUGCGGUUGUGCTsT 34% 3% ND-10759 1237 ACUGCUUCUACCAGACAUATsT 1238 UAUGUCUGGUAGAAGCAGUTsT 11% 1% ND-10760 1239 GAAGACGGCCUUCUGGGCATsT 1240 UGCCCAGAAGGCCGUCUUCTsT 16% 2% ND-10761 1241 AAGACGGCCUUCUGGGCAGTsT 1242 CUGCCCAGAAGGCCGUCUUTsT 58% 21%  ND-10762 1243 ACAUCAACCUCAACUCGGATsT 1244 UCCGAGUUGAGGUUGAUGUTsT 14% 3% ND-10763 1245 UGGAAGGACUGGAAGAUCGTsT 1246 CGAUCUUCCAGUCCUUCCATsT 109%  29%  ND-10764 1247 ACAUCCUGUCGAGGCUGCCTsT 1248 GGCAGCCUCGACAGGAUGUTsT 101%  13%  ND-10765 1249 CAACCAGGCGAAUUACUCUTsT 1250 AGAGUAAUUCGCCUGGUUGTsT 19% 5% ND-10766 1251 CAGGCGAAUUACUCUCACUTsT 1252 AGUGAGAGUAAUUCGCCUGTsT 24% 4% ND-10767 1253 AGCAGAAUGACUUCAUUCCTsT 1254 GGAAUGAAGUCAUUCUGCUTsT 40% 8% ND-10768 1255 AUGAUGGUGGCUUUAACUUTsT 1256 AAGUUAAAGCCACCAUCAUTsT 85% 8% ND-10769 1257 AGAACCUUUACCCUUCAAATsT 1258 UUUGAAGGGUAAAGGUUCUTsT 22% 4% ND-10770 1259 CCUUUACCCUUCAAAGUACTsT 1260 GUACUUUGAAGGGUAAAGGTsT 21% 4% ND-10771 1261 GAGCCUGUGGUUCGGCUCCTsT 1262 GGAGCCGAACCACAGGCUCTsT 28% 1% ND-10772 1263 UGGUACUGCCUCUGAACACTsT 1264 GUGUUCAGAGGCAGUACCATsT 58% 4% ND-10773 1265 CUCAUACCCGUGCCCUCACTsT 1266 GUGAGGGCACGGGUAUGAGTsT 15% 2% ND-10774 1267 CCGUAGCACACUAUAACAUTsT 1268 AUGUUAUAGUGUGCUACGGTsT 24% 6% ND-10775 1269 CGUAGCACACUAUAACAUCTsT 1270 GAUGUUAUAGUGUGCUACGTsT 21% 4% ND-10776 1271 GCAGGACCCUAGACCUCUGTsT 1272 CAGAGGUCUAGGGUCCUGCTsT 25% 4% ND-10777 1273 GCCUGCUUUUCGGAGAGUATsT 1274 UACUCUCCGAAAAGCAGGCTsT 18% 4% ND-10778 1275 GGGCCCGGGUAAUGGUGCATsT 1276 UGCACCAUUACCCGGGCCCTsT 16% 2% ND-10779 1277 CAACAACAAGAGAAAUGGATsT 1278 UCCAUUUCUCUUGUUGUUGTsT 17% 0% ND-10780 1279 GCUGUUGCACCAUACUUUCTsT 1280 GAAAGUAUGGUGCAACAGCTsT 14% 1% ND-10781 1281 CUACCGAGAGCUCUUCGAGTsT 1282 CUCGAAGAGCUCUCGGUAGTsT 24% 3% ND-10782 1283 ACCUGCCUUUAUGGAUGAUTsT 1284 AUCAUCCAUAAAGGCAGGUTsT 115%  10%  ND-10783 1285 UUGACAAGGAACUUUCCUATsT 1286 UAGGAAAGUUCCUUGUCAATsT 16% 1% ND-10784 1287 GCUGGAGUGUUGCUGUUGCTsT 1288 GCAACAGCAACACUCCAGCTsT 12% 1% ND-10785 1289 UCGGUGACAUCCCAGGAAUTsT 1290 AUUCCUGGGAUGUCACCGATsT 2

% 1% ND-10786 1291 GCUGCCCAGAAGUGCCUUGTsT 1292 CAAGGCACUUCUGGGCAGCTsT 15% 2% ND-10787 1293 AGUACACACAGCAGGUGUGTsT 1294 CACACCUGCUGUGUGUACUTsT 12% 1% ND-10788 1295 CAAGUGCCGGAAGCCAUGCTsT 1296 GCAUGGCUUCCGGCACUUGTsT 94% 2%

indicates data missing or illegible when filed

TABLE 1C Selected siRNAs in in vivo rat surrogate set (human-rat cross-reactive siRNAs with highest specificity in rat). A screening set of 48 human and rat cross-reactive alpha-ENaC iRNA sequences were identified. The percentage residual expression of alpha-ENaC in two independent single-dose transfection experiments is shown (refer to examples section for methods used). 1st screen  single dose @ 50 nM 2nd in screen @ Seq H441; 50 nM in Duplex ID Seq ID Sense ID Antissense MV SD H441 SD ND-9201 1297 uGuGcAAccAGAAcAAAucTsT 1298 GAUUUGUUCUGGUUGcAcATsT  8% 1%  8% 1% ND-9202 1299 uuuAuGGAuGAuGGuGGcuTsT 1300 AGCcACcAUcAUCcAuAAATsT 80% 9% 82% 6% ND-9203 1301 GccuuuAuGGAuGAuGGuGTsT 1302 cACcAUcAUCcAuAAAGGCTsT 76% 8% 76% 2% ND-9204 1303 cAcAAccGcAuGAAGAcGGTsT 1304 CCGUCUUcAUGCGGUUGUGTsT 73% 18%  57% 3% ND-9205 1305 AccGcAuGAAGAcGGccuuTsT 1306 AAGGCCGUCUUcAUGCGGUTsT 355 3% 37% 2% ND-9206 1307 AGGAcuGGAAGAucGGcuuTsT 1308 AAGCCGAUCUUCcAGUCCUTsT 17% 3% 16% 3% ND-9207 1309 GAAGGAcuGGAAGAucGGcTsT 1310 GCCGAUCUUCcAGUCCUUCTsT 96% 18%  81% 5% ND-9208 1311 GGAcuGGAAGAucGGcuucTsT 1312 GAAGCCGAUCUUCcAGUCCTsT 58% 6%

7% 3% ND-9209 1313 AGuuccAccGcuccuAccGTsT 1314 CGGuAGGAGCGGUGGAACUTsT 85% 8% 94% 4% ND-9210 1315 GAcuGGAAGAucGGcuuccTsT 1316 GGAAGCCGAUCUUCcAGUCTsT 79% 5% 82% 2% ND-9211 1317 cGcAuGAAGAcGGccuucuTsT 1318 AGAAGGCCGUCUUcAUGCGTsT

0% 1% 51% 1% ND-9212 1319 GccAGuGGAGccuGuGGuuTsT 1320 AACcAcAGGCUCcACUGGCTsT 26% 3% 23% 2% ND-9213 1321 uGccuuuAuGGAuGAuGGuTsT 1322 ACcAUcAUCcAuAAAGGcATsT 77% 5% 76% 4% ND-9214 1323 uccuGuccAAccuGGGcAGTsT 1324 CUGCCcAGGUUGGAcAGGATsT 74% 9% 83% 6% ND-9215 1325 AGGGAGuGGuAccGcuuccTsT 1326 GGAAGCGGuACcACUCCCUTsT 79% 6% 89% 4% ND-9216 1327 GGcuGuGccuAcAucuucuTsT 1328 AGAAGAUGuAGGcAcAGCCTsT 11% 1% 13% 1% ND-9217 1329 GAAAuuAAAGAGGAGcuGGTsT 1330 CcAGCUCCUCUUuAAUUUCTsT 84% 14%  78% 5% ND-9218 1331 AcuGGAAGAucGGcuuccATsT 1332 UGGAAGCCGAUCUUCcAGUTsT 50% 4% 55% 3% ND-9219 1333 ccuGuccAAccuGGGcAGcTsT 1334 GCUGCCcAGGUUGGAcAGGTsT 78% 6% 85% 5% ND-9220 1335 ccuGccuuuAuGGAuGAuGTsT 1336 cAUcAUCcAuAAAGGcAGGTsT 76% 5% 77% 9% ND-9221 1337 AAccGcAuGAAGAcGGccuTsT 1338 AGGCCGUCUUcAUGCGGUUTsT 79% 8% 66% 3% ND-9222 1339 uGuccAAccuGGGcAGccATsT 1340 UGGCUGCCcAGGUUGGAcATsT 70% 4% 57% 4% ND-9223 1341 GuccAAccuGGGcAGccAGTsT 1342 CUGGCUGCCcAGGUUGGACTsT 9

% 10%  76% 4% ND-9224 1343 AAAuuAAAGAGGAGcuGGATsT 1344 UCcAGCUCCUCUUuAAUUUTsT 83% 6% 69% 2% ND-9225 1345 GGAAGGAcuGGAAGAucGGTsT 1346 CCGAUCUUCcAGUCCUUCCTsT 41% 2% 30% 2% ND-9226 1347 GuGAGGGAGuGGuAccGcuTsT 1348 AGCGGuACcACUCCCUcACTsT 21% 1% 17%

% ND-9227 1349 AcuuucAAuGAcAAGAAcATsT 1350 UGUUCUUGUcAUUGAAAGUTsT 13% 1% 10%

% ND-9228 1351 ucAAuGAcAAGAAcAAcucTsT 1352 GAGUUGUUCUUGUcAUUGATsT 36% 2% 28%

% ND-9229 1353 cuuuAuGGAuGAuGGuGGcTsT 1354 GCcACcAUcAUCcAuAAAGTsT 24% 1% 20% 1% ND-9230 1355 GccuGGcGuGGAGAccuccTsT 1356 GGAGGUCUCcACGCcAGGCTsT ND-9231 1357 uGGcGuGGAGAccuccAucTsT 1358 GAUGGAGGUCUCcACGCcATsT 45% 4% 35% 2% ND-9232 1359 GAGuuccAccGcuccuAccTsT 1360 GGuAGGAGCGGUGGAACUCTsT 89% 4% 86% 8% ND-9233 1361 cAGAGcAGAAuGAcuucAuTsT 1362 AUGAAGUcAUUCUGCUGUGTsT 21% 1% 17% 8% ND-9234 1363 uucAcuccuGcuuccAGGATsT 1364 UCCUGGAAGcAGGAGUGAATsT 85% 4% 74% 6% ND-9235 1365 ucAcuccuGcuuccAGGAGTsT 1366 CUCCUGGAAGcAGGAGUGATsT ND-9236 1367 cuGuGcAAccAGAAcAAAuTsT 1368 AUUUGUUCUGGUUGcAcAGTsT 23% 1% 17% 2% ND-9237 1369 cuGcAAcAAcAccAccAucTsT 1370 GAUGGUGGUGUUGUUGcAGTsT 34% 2% 27% 2% ND-9238 1371 uGuGGcuGuGccuAcAucuTsT 1372 AGAUGuAGGcAcAGCcAcATsT 86% 4% 73% 10%  ND-9239 1373 uGGcuGuGccuAcAucuucTsT 1374 GAAGAUGuAGGcAcAGCcATsT 68% 6% 53% 4% ND-9240 1375 cuGuccAAccuGGGcAGccTsT 1376 GGCUGCCcAGGUUGGAcAGTsT 80% 5% 73% 9% ND-9241 1377 cccuGcuGuccAcAGuGAcTsT 1378 GUcACUGUGGAcAGcAGGGTsT 83%

% 71% 5% ND-9242 1379 GcAGccAGuGGAGccuGUGTsT 1380 cAcAGGCUCcACUGGCUGCTsT 105% 9% 90% 5% ND-9243 1381 uucAAuGAcAAGAAcAAcuTsT 1382 AGUUGUUCUUGUcAUUGAATsT 23% 3% 21% 1% ND-9244 1383 cuGccuuuAuGGAuGAuGGTsT 1384 CcAUcAUCcAuAAAGGcAGTsT 74% 6% 64% 7% ND-9245 1385 AAuGAcAAGAAcAAcuccATsT 1386 UGGAGUUGUUCUUGUcAUUTsT 21% 1% 21% 1% ND-9246 1387 uGGGcAGccAGuGGAGccuTsT 1388 AGGCUCcACUGGCUGCCcATsT 83% 3% 73% 2% ND-9247 1389 cuccuGuccAAccuGGGcATsT 1390 UGCCcAGGUUGGAcAGGAGTsT 86% 3% 84% 1% ND-9248 1391 GGcGuGGAGAccuccAucATsT 1392 UGAUGGAGGUCUCcACGUCTsT 92% 4% 88% 3%

indicates data missing or illegible when filed

TABLE 1D Selected siRNAs in in vivo guinea pig surrogate set (human-guinea pig cross-reactive siRNAs). A screening set of 63 human and guinea-pig cross-reactive alpha-ENaC iRNA sequences were identified and synthesised, both with (sequence strands 1393-1518) and without (sequence strands 1519-1644) backbone modification. The percentage residual expression of alpha-ENaC in two independent single-dose transfection experiments is shown (refer to examples section for methods used). 1st screen single dose @ 50 nM 2nd in screen @ H441; 50 nM in Duplex ID Seq ID Sense Seq ID Antissense MV SD H441 SD ND8437 1393 AAucGGAcuGcuucuAccATsT 1394 UGGuAGAAGcAGUCCGAUUTsT 4

% 7% 46% 5% ND8438 1395 AucGGAcuGcuucuAccAGTsT 1396 CUGGuAGAAGcAGUCCGAUTsT 85% 5% 93% 13%  ND8439 1397 AAAucGGAcuGcuucuAccTsT 1398 GGuAGAAGcAGUCCGAUUUTsT 36% 3% 42% 6% ND8440 1399 ucGGAcuGcuucuAccAGATsT 1400 UCUGGuAGAAGcAGUCCGATsT 45% 3% 50% 4% ND8441 1401 AccAGAAcAAAucGGAcuGTsT 1402 cAGUCCGAUUUGUUCUGGUTsT 23% 3% 24% 6% ND8442 1403 ccAGAAcAAAucGGAcuGcTsT 1404 GcAGUCCGAUUUGUUCUGGTsT 50% 6% 39% 9% ND8443 1405 cAGAAcAAAucGGAcuGcuTsT 1406 AGcAGUCCGAUUUGUUCUGTsT 22%

%

4% 1% ND8444 1407 cuucGccuGccGcuucAAcTsT 1408 GUUGAAGCGGcAGGCGAAGTsT 111%  8% 109%  4% ND8445 1409 uGGuAccGcuuccAcuAcATsT 1410 UGuAGUGGAAGCGGuACcATsT 84% 7% 97% 13%  ND8446 1411 AucuucGccuGccGcuucATsT 1412 UGAAGCGGcAGCGCGAAGUTsT 90% 3% 121%  13%  ND8447 1413 uucGccuGccGcuucAAccTsT 1414 GGUUGAAGCGGcAGGCGAATsT 92% 2% 105%  17%  ND8448 1415 cAcccucAAucccuAcAGGTsT 1416 CCUGuAGGGAUUGAGGGUGTsT 79% 3% 90% 13%  ND8449 1417 AGAAcAAAucGGAcuGcuuTsT 1418 AAGcAGUCCGAUUUGUUCUTsT 11% 0% 17% 3% ND8450 1419 GAAcAAAucGGAcuGcuucTsT 1420 GAAGcAGUCCGAUUUGUUCTsT 21% 1% 30% 5% ND8451 1421 cGGAcuGcuucuAccAGAcTsT 1422 GUCUGGuAGAAGcAGUCCGTsT 24% 2% 32% 5% ND8452 1423 AGccucAAcAucAAccucATsT 1424 UGAGGUUGAUGUUGAGGCUTsT 51% 3% 57% 4% ND8453 1425 GccucAAcAucAAccucAATsT 1426 UUGAGGUUGAUGUUGAGGCTsT 16% 1% 26% 3% ND8454 1427 GucAGccucAAcAucAAccTsT 1428 GGUUGAUGUUGAGGCUGACTsT 62% 5% 68% 6% ND8455 1429 ucAGccucAAcAucAAccuTsT 1430 AGGUUGAUGUUGAGGCUGATsT 77% 4% 87% 6% ND8456 1431 cAGccucAAcAucAAccucTsT 1432 GAGGUUGAUGUUGAGGCUGTsT 34% 2% 51% 8% ND8457 1433 GGAGcuGGAccGcAucAcATsT 1434 UGUGAUGCGGUCcAGCUCCTsT 26% 2% 17% 1% ND8458 1435 GuAccGcuuccAcuAcAucTsT 1436 GAUGuAGUGGAAGCGGuACTsT 101%  9% 99% 11%  ND8459 1437 ccGcuuccAcuAcAucAAcTsT 1438 GUUGAUGuAGUGGAAGCGGTsT 85% 8% 80% 6% ND8460 1439 cGcuuccAcuAcAucAAcATsT 1440 UGUUGAUGuAGUGGAAGCGTsT 56% 6% 48% 3% ND8461 1441 uuccAcuAcAucAAcAuccTsT 1442 GGAUGUUGAUGuAGUGGAATsT 77% 5% 82% 7% ND8462 1443 uGGGcAAcuucAucuucGcTsT 1444 GCGAAGAUGAAGUUGCCcATsT 21% 0% 36% 6% ND8463 1445 GcAAcuucAucuucGccuGTsT 1446 cAGGCGAAGAUGAAGUUGCTsT 80% 4% 84% 13%  ND8464 1447 cAAcuucAucuucGccuGcTsT 1448 GcAGGCGAAGAUGAAGUUGTsT 101%  1% 102%  14%  ND8465 1449 AAcuucAucuucGccuGccTsT 1450 GGcAGGCGAAGAUGAAGUUTsT 100%  4% 95% 12%  ND8466 1451 AcuucAucuucGccuGccGTsT 1452 CGGcAGGCGAAGAUGAAGUTsT 51% 4% 49% 5% ND8467 1453 cuucAucuucGccuGccGcTsT 1454 GCGGcAGGCGAAGAUGAAGTsT 95% 5% 89% 4% ND8468 1455 ucAucuucGccuGccGcuuTsT 1456 AAGCGGcAGGCGAAGAUGATsT 91% 4% 85% 6% ND8469 1457 cAucuucGccuGccGcuucTsT 1458 GAAGCGGcAGGCGAAGAUGTsT 66% 4% 55% 4% ND8470 1459 ucuucGccuGccGcuucAATsT 1460 UUGAAGCGGcAGGCGAAGATsT 97% 2% 99% 11%  ND8471 1461 cGccuGccGcuucAAccAGTsT 1462 CUGGUUGAAGCGGcAGGCGTsT 96% 4% 100%  7% ND8472 1463 GccuGccGcuucAAccAGGTsT 1464 CCUGGUUGAAGCGGcAGGCTsT 90% 4% 82% 5% ND8473 1465 AuuAcucucAcuuccAccATsT 1466 UGGUGGAAGUGAGAGuAAUTsT 81% 3% 72% 4% ND8474 1467 uuAcucucAcuuccAccAcTsT 1468 GUGGUGGAAGUGAGAGuAATsT 72% 2% 76% 9% ND8475 1469 AcucucAcuuccAccAcccTsT 1470 GGGUGGUGGAAGUGAGAGUTsT 90% 3% 97% 4% ND8476 1471 ucuGcAcccucAAucccuATsT 1472 uAGGGAUUGAGGGUGcAGATsT 61% 1% 63% 3% ND8477 1473 cuGcAcccucAAucccuAcTsT 1474 GuAGGGAUUGAGGGUGcAGTsT 74% 3% 73% 1% ND8478 1475 uGcAcccucAAucccuAcATsT 1476 UGuAGGGAUUGAGGGUGcATsT 98% 4% 85% 1% ND8479 1477 AcccucAAucccuAcAGGuTsT 1478 ACCUGuAGGGAUUGAGGGUTsT 55% 5% 48% 3% ND8480 1479 cccucAAucccuAcAGGuATsT 1480 uACCUGuAGGGAUUGAGGGTsT 20% 1% 14% 1% ND8481 1481 ccucAAucccuAcAGGuAcTsT 1482 GuACCUGuAGGGAUUGAGGTsT 40% 2% 31% 3% ND8482 1483 AAccAGAAcAAAucGGAcuTsT 1484 AGUCCGAUUUGUUCUGGUUTsT 57% 2% 52% 0% ND8483 1485 AAcAAAucGGAcuGcuucuTsT 1486 AGAAGcAGUCCGAUUUGUUTsT 102%  5% 86% 12%  ND8484 1487 AcAAAucGGAcuGcuucuATsT 1488 uAGAAGcAGUCCGAUUUGUTsT 40% 2% 28% 3% ND8485 1489 cAAAucGGAcuGcuucuAcTsT 1490 GuAGAAGcAGUCCGAUUUGTsT 41% 4% 38% 2% ND8486 1491 GcAcccucAAucccuAcAGTsT 1492 CUGuAGGGAUUGAGGGUGCTsT 91% 7% 94% 4% ND8487 1493 ccucAAcAucAAccucAAcTsT 1494 GUUGAGGUUGAUGUUGAGGTsT 46% 2% 37% 3% ND8488 1495 cucAAcAucAAccucAAcuTsT 1496 AGUUGAGGUUGAUGUUGAGTsT 48% 2% 39% 3% ND8489 1497 ucAAcAucAAccucAAcucTsT 1498 GAGUUGAGGUUGAUGUUGATsT 17% 1% 17% 1% ND8490 1499 uAccGcuuccAcuAcAucATsT 1500 UGAUGuAGUGGAAGCGGuATsT 90% 5% 74% 8% ND8491 1501 AccGcuuccAcuAcAucAATsT 1502 UUGAUGuAGUGGAAGCGGUTsT 103%  5% 91% 15%  ND8492 1503 GcuuccAcuAcAucAAcAuTsT 1504 AUGUUGAUGuAGUGGAAGCTsT 85% 5% 71% 10%  ND8493 1505 cuuccAcuAcAucAAcAucTsT 1506 GAUGUUGAUGuAGUGGAAGTsT 60% 5% 45% 3% ND8494 1507 uccAcuAcAucAAcAuccuTsT 1508 AGGAUGUUGAUGuAGUGGATsT 33% 3% 41% 3% ND8495 1509 ccAcuAcAucAAcAuccuGTsT 1510 cAGGAUGUUGAUGuAGUGGTsT 60% 5% 55% 2% ND8496 1511 cuGGGcAAcuucAucuucGTsT 1512 CGAAGAUGAAGUUGCCcAGTsT 18% 0% 20% 0% ND8497 1513 GGcAAcuucAucuucGccuTsT 1514 AGGCGAAGAUGAAGUUGCCTsT 76% 1% 77% 2% ND8498 1515 uucAucuucGccuGccGcuTsT 1516 AGCGGcAGGCGAAGAUGAATsT 65% 4% 74% 12%  ND8499 1517 ucGccuGccGcuucAAccATsT 1518 UGGUUGAAGCGGcAGGCGATsT 86% 5% 77% 3% ND-8653 1519 AAUCGGACUGCUUCUACCATsT 1520 UGGUAGAAGCAGUCCGAUUTsT 16% 2% 20% 3% ND-8654 1521 AUCGGACUGCUUCUACCAGTsT 1522 CUGGUAGAAGCAGUCCGAUTsT 54% 8% 67% 11%  ND-8655 1523 AAAUCGGACUGCUUCUACCTsT 1524 GGUAGAAGCAGUCCGAUUUTsT 25% 4% 28% 2% ND-8656 1525 UCGGACUGCUUCUACCAGATsT 1526 UCUGGUAGAAGCAGUCCGATsT 12% 2% 17% 1% ND-8657 1527 ACCAGAACAAUCGGGACUGTsT 1528 CAGUCCGAUUUGUUCUGGUTsT 33% 3% 35% 1% ND-8658 1529 CCAGAACAAAUCGGACUGCTsT 1530 GCAGUCCGAUUUGUUCUGGTsT 27% 3% 30% 2% ND-8659 1531 CAGAACAAAUCGGACUGCUTsT 1532 AGCAGUCCGAUUUGUUCUGTsT 15% 1% 22% 3% ND-8660 1533 CUUCGCCUGCCGCUUCAACTsT 1534 GUUGAAGCGGCAGGCGAAGTsT 69% 17%  75% 10%  ND-8661 1535 UGGUACCGCUUCCACUACATsT 1536 UGUAGUGGAAGCGGUACCATsT 16% 2% 20% 3% ND-8662 1537 AUCUUCGCCUGCCGCUUCATsT 1538 UGAAGCGGCAGGCGAAGAUTsT 19% 2% 25% 4% ND-8663 1539 UUCGCCUGCCGCUUCAACCTsT 1540 GGUUGAAGCGGCAGGCGAATsT 90% 4% 97% 10%  ND-8664 1541 CACCCUCAAUCCCUACAGGTsT 1542 CCUGUAGGGAUUGAGGGUGTsT 19% 2% 25% 3% ND-8665 1543 AGAACAAAUCGGACUGCUUTsT 1544 AAGCAGUCCGAUUUGUUCUTsT 13% 1% 22% 2% ND-8666 1545 GAACAAAUCGGACUGCUUCTsT 1546 GAAGCAGUCCGAUUUGUUCTsT 11% 2% 18% 2% ND-8667 1547 CGGACUGCUUCUACCAGACTsT 1548 GUCUGGUAGAAGCAGUCCGTsT 13% 1% 16% 2% ND-8668 1549 AGCCUCAACAUCAACCUCATsT 1550 UGAGGUUGAUGUUGAGGCUTsT 17% 4% 21% 3% ND-8669 1551 GCCUCAACAUCAACCUCAATsT 1552 UUGAGGUUGAUGUUGAGGCTsT 13% 1% 21% 3% ND-8670 1553 GUCAGCCUCAACAUCAACCTsT 1554 GGUUGAUGUUGAGGCUGACTsT 43% 11%  27% 3% ND-8671 1555 UCAGCCUCAACAUCAACCUTsT 1556 AGGUUGAUGUUGAGGCUGATsT 90% 17%  53% 13%  ND-8672 1557 CAGCCUCAACAUCAACCUCTsT 1558 GAGGUUGAUGUUGAGGCUGTsT 17% 3% 11% 3% ND-8673 1559 GGAGCUGGACCGCAUCACATsT 1560 UGUGAUGCGGUCCAGCUCCTsT 25% 3% 18% 3% ND-8674 1561 GUACCGCUUCCACUACAUCTsT 1562 GAUGUAGUGGAAGCGGUACTsT 21% 4% 16% 4% ND-8675 1563 CCGCUUCCACUACAUCAACTsT 1564 GUUGAUGUAGUGGAAGCGGTsT 25% 4% 19% 3% ND-8676 1565 CGCUUCCACUACAUCAACATsT 1566 UGUUGAUGUAGUGGAAGCGTsT 16% 3% 14% 1% ND-8677 1567 UUCCACUACAUCAACAUCCTsT 1568 GGAUGUUGAUGUAGUGGAATsT 110%  19%  97% 9% ND-8678 1569 UGGGCAACUUCAUCUUCGCTsT 1570 GCGAAGAUGAAGUUGCCCATsT 50% 8% 40% 5% ND-8679 1571 GCAACUUCAUCUUCGCCUGTsT 1572 CAGGCGAAGAUGAAGUUGCTsT 19% 3% 17% 2% ND-8680 1573 CAACUUCAUCUUCGCCUGCTsT 1574 GCAGGCGAAGAUGAAGUUGTsT 25% 2% 23% 2% ND-8681 1575 AACUUCAUCUUCGCCUGCCTsT 1576 GGCAGGCGAAGAUGAAGUUTsT 104%  7% 85% 10%  ND-8682 1577 ACUUCAUCUUCGCCUGCCGTsT 1578 CGGCAGGCGAAGAUGAAGUTsT 91% 8% 63% 9% ND-8683 1579 CUUCAUCUUCGCCUGCCGCTsT 1580 GCGGCAGGCGAAGAUGAAGTsT 88% 6% 58% 6% ND-8684 1581 UCAUCUUCGCCUGCCGCUUTsT 1582 AAGCGGCAGGCGAAGAUGATsT 76% 3% 64% 4% ND-8685 1583 CAUCUUCGCCUGCCGCUUCTsT 1584 GAAGCGGCAGGCGAAGAUGTsT 15% 1% 18% 3% ND-8686 1585 UCUUCGCCUGCCGCUUCAATsT 1586 UUGAAGCGGCAGGCGAAGATsT 109%  22%  31% 3% ND-8687 1587 CGCCUGCCGCUUCAACCAGTsT 1588 CUGGUUGAAGCGGCAGGCGTsT 90% 21%  49% 2% ND-8688 1589 GCCUGCCGCUUCAACCAGGTsT 1590 CCUGGUUGAAGCGGCAGGCTsT 43% 9% 24% 7% ND-8689 1591 AUUACUCUCACUUCCACCATsT 1592 UGGUGGAAGUGAGAGUAAUTsT 27% 4% 19% 2% ND-8690 1593 UUACUCUCACUUCCACCACTsT 1594 GUGGUGGAAGUGAGAGUAATsT 109%  7% 85% 8% ND-8691 1595 ACUCUCACUUCCACCACCCTsT 1596 GGGUGGUGGAAGUGAGAGUTsT 93% 11%  87% 12%  ND-8692 1597 UCUGCACCCUCAAUCCCUATsT 1598 UAGGGAUUGAGGGUGCAGATsT 31% 12%  17% 2% ND-8693 1599 CUGCACCCUCAAUCCCUACTsT 1600 GUAGGGAUUGAGGGUGCAGTsT 41% 25%  31% 4% ND-8694 1601 UGCACCCUCAAUCCCUACATsT 1602 UGUAGGGAUUGAGGGUGCATsT 75% 25%  43% 3% ND-8695 1603 ACCCUCAAUCCCUACAGGUTsT 1604 ACCUGUAGGGAUUGAGGGUTsT 65% 26%  25% 5% ND-8696 1605 CCCUCAAUCCCUACAGGUATsT 1606 UACCUGUAGGGAUUGAGGGTsT 18% 2% 13% 1% ND-8697 1607 CCUCAAUCCCUACAGGUACTsT 1608 GUACCUGUAGGGAUUGAGGTsT 16% 4% 13% 2% ND-8698 1609 AACCAGAACAAAUCGGACUTsT 1610 AGUCCGAUUUGUUCUGGUUTsT 40% 2% 30% 2% ND-8699 1611 AACAAAUCGGACUGCUUCUTsT 1612 AGAAGCAGUCCGAUUUGUUTsT 56% 4% 45% 3% ND-8700 1613 ACAAAUCGGACUGCUUCUATsT 1614 UAGAAGCAGUCCGAUUUGUTsT 18% 3% 12% 1% ND-8701 1615 CAAAUCGGACUGCUUCUACTsT 1616 GUAGAAGCAGUCCGAUUUGTsT 15% 2% 15% 4% ND-8702 1617 GCACCCUCAAUCCCUACAGTsT 1618 CUGUAGGGAUUGAGGGUGCTsT 53% 4% 46% 20%  ND-8703 1619 CCUCAACAUCAACCUCAACTsT 1620 GUUGAGGUUGAUGUUGAGGTsT 25% 6% 26% 9% ND-8704 1621 CUCAACAUCAACCUCAACUTsT 1622 AGUUGAGGUUGAUGUUGAGTsT 30% 8% 37% 26%  ND-8705 1623 UCAACAUCAACCUCAACUCTsT 1624 GAGUUGAGGUUGAUGUUGATsT 55% 1% 50% 10%  ND-8706 1625 UACCGCUUCCACUACAUCATsT 1626 UGAUGUAGUGGAAGCGGUATsT 36% 7% 31% 7% ND-8707 1627 ACCGCUUCCACUACAUCAATsT 1628 UUGAUGUAGUGGAAGCGGUTsT 23% 5% 27% 10%  ND-8708 1629 GCUUCCACUACAUCAACAUTsT 1630 AUGUUGAUGUAGUGGAAGCTsT 16% 4% 24% 12%  ND-8709 1631 CUUCCACUACAUCAACAUCTsT 1632 GAUGUUGAUGUAGUGGAAGTsT 62% 3% 74% 27%  ND-8710 1633 UCCACUACAUCAACAUCCUTsT 1634 AGGAUGUUGAUGUAGUGGATsT 45% 8% 41% 1% ND-8711 1635 CCACUACAUCAACAUCCUGTsT 1636 CAGGAUGUUGAUGUAGUGGTsT 23% 4% 27% 10%  ND-8712 1637 CUGGGCAACUUCAUCUUCGTsT 1638 CGAAGAUGAAGUUGCCCAGTsT 34% 4% 26% 5% ND-8713 1639 GGCAACUUCAUCUUCGCCUTsT 1640 AGGCGAAGAUGAAGUUGCCTsT 30% 3% 23% 2% ND-8714 1641 UUCAUCUUCGCCUGCCGCUTsT 1642 AGCGGCAGGCGAAGAUGAATsT 90% 14%  85% 14%  ND-8715 1643 UCGCCUGCCGCUUCAACCATsT 1644 UGGUUGAAGCGGCAGGCGATsT 23% 2% 20% 4%

indicates data missing or illegible when filed

TABLE 2A Concentration at 50% inhibition (IC50) for exemplary iRNA agents of Table 1A IC50 [nM] 1st IC50 [nM] 2nd Duplex ID DRC in H441 DRC in H441 ND8294 0.1949 0.0468 ND8295 0.1011 0.0458 ND8299 0.5986 0.5638 ND8302 0.0144 0.0134 ND8313 0.0315 0.0124 ND8320 0.0796 0.0078 ND8331 0.0213 0.0158 ND8332 0.0205 0.0089 ND8343 0.0523 0.0293 ND8348 0.0156 0.0182 ND8356 0.0241 0.0099 ND8357 0.0054 0.0032 ND8363 0.1186 0.0337 ND8368 0.0487 0.1209 ND8371 0.0811 0.0911 ND8372 0.0584 0.0425 ND8373 0.0066 0.0165 ND8375 0.1176 0.1187 ND8380 0.6817 0.5747 ND8381 0.0037 0.0041 ND8383 0.0275 0.1257 ND8384 0.0357 0.0082 ND8391 0.0260 0.0349 ND8392 0.3831 0.4775 ND8396 0.0023 0.0052 ND8403 0.0808 0.0759

TABLE 2B Concentration at 50% inhibition (IC50) and for exemplary iRNA agents of Table 1D. IC50 [nM] 1st IC50 [nM] 2nd Duplex ID DRC in H441 DRC in H441 ND8441 0.6738 0.8080 ND8443 0.0346 0.0263 ND8449 0.0120 0.0067 ND8450 0.0257 0.0106 ND8451 0.1320 0.0931 ND8453 0.0079 0.0033 ND8489 0.1640 0.1593 ND8496 0.0387 0.0185

TABLE 2C % Activity of the exemplary RNAi towards inhibition of alpha- ENaC gene expression in the assays described in Example 3 % alpha-ENaC Cynomolgous alpha- expression in primary ENaC expression HBEC (% of control) (% of control) Duplex identifier 50 nM siRNA 45 nM siRNA Untransfected 77.2 n/a Non-targetting Control 100 93.3 Negative Control n/a 100 (Non-cyno alpha-ENaC) ND8449 ND-8302 30.2 57 ND-8332 24.7 54.3 ND-8348 40.1 56.2 ND-8356 36.6 55.8 ND-8357 29.6 50.4 ND-8373 30.4 53.8 ND-8381 32.5 40.4 ND-8396 34.1 46.3 ND-8450 45.9 78.9 ND-8453 30.1 55.3 

1. A composition comprising an iRNA agent for inhibiting the expression of an alpha-ENaC gene, wherein the iRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence of nucleotides 2-18 of SEQ ID NO: 980, SEQ ID NO: 1298, or SEQ ID NO:
 1368. 2. The composition of claim 1, wherein the sense strand of the iRNA agent comprises a nucleotide sequence of nucleotides 2-18 of SEQ ID NO: 979, SEQ ID NO: 1297, or SEQ ID NO:
 1367. 3. The composition of claim 1, wherein the antisense strand of the iRNA agent and the sense strand of the iRNA agent are each 19 to 23 nucleotides in length.
 4. The composition of claim 1, wherein the iRNA agent comprises at least one modified nucleotide and/or at least one phosphate linker modification.
 5. The composition of claim 2, wherein the iRNA agent comprises at least on modified nucleotide and/or at least one phosphate linker modification.
 6. The composition of claim 4, wherein the iRNA agent comprises one or more 2′-modified nucleotides and/or one or more phosphorothioates.
 7. The composition of claim 5, wherein the iRNA agent comprises one or more 2′-modified nucleotides and/or one or more phosphorothioates.
 8. The composition of claim 6, wherein the one or more 2′-modified nucleotides are independently selected from the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).
 9. The composition of claim 7, wherein the one or more 2′-modified nucleotides are independently selected from the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).
 10. The composition of claim 4, wherein the sense strand and/or the antisense strand contains a 3′ overhang.
 11. The composition of claim 4, wherein the sense strand and/or the antisense strand contains a 5′ overhang.
 12. The composition of claim 4, wherein the iRNA agent comprises at least one blunt end.
 13. The composition of claim 4, further comprising a buffer, diluent, penetration enhancer, carrier compound, and/or pharmaceutically acceptable carrier or excipient.
 14. The composition of claim 4, wherein the composition is presented in unit dosage form.
 15. The composition of claim 1, wherein the iRNA agent is ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, Oligo Lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, epithelial receptor ligand, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.
 16. The composition of claim 4, further comprising one or more known agents effective in treatment of ENaC-related disorders.
 17. The composition of claim 16, wherein the known agent is selected from the group consisting of: anti-inflammatory drug, bronchodilatory drug, antihistamine, anti-tussive drug, antibiotic, DNase drug substance, epithelial sodium channel blocker.
 18. The composition of claim 5, wherein the iRNA agent comprises: a) an antisense strand comprising a nucleotide sequence of nucleotides 1-19 of SEQ ID NO: 980, and a sense strand comprising a nucleotide sequence of nucleotides 1-19 of SEQ ID NO: 979, b) an antisense strand comprising a nucleotide sequence of nucleotides 1-19 of SEQ ID NO: 1298 and a sense strand comprising a nucleotide sequence of nucleotides 1-19 of SEQ ID NO: 1297, or c) an antisense strand comprising a nucleotide sequence of nucleotides 1-19 of SEQ ID NO: 1368, and a sense strand comprising a nucleotide sequence of nucleotides 1-19 of SEQ ID NO:
 1367. 19. A method of treating a human subject having a pathological state mediated at least in part by alpha-ENaC expression, the method comprising the step of administering to the subject a therapeutically effective amount of a composition of claim
 4. 20. The method of claim 19, wherein the pathological state is cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, respiratory tract infections, lung carcinoma, Liddles syndrome, hypertension, renal insufficiency, and/or electrolyte imbalance.
 21. The method of claim 19, wherein the composition is administered via inhalation/intranasal administration or systemically or subcutaneously.
 22. A method of inhibiting the expression of an alpha-ENaC gene in a cell, the method comprising administering to the cell an effective amount of a composition of claim
 4. 23. A composition comprising an iRNA agent comprising a sense strand and an antisense strand, wherein: the antisense strand comprises a nucleotide sequence of nucleotides 2-18 of any of the antisense strands Table 1A, Table 1B, Table 1C, or Table 1D. 